Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1

Muscle tissue is the major site for insulin-stimulated glucose uptake in vivo, due primarily to the recruitment of the insulin-sensitive glucose transporter (GLUT4) to the plasma membrane. Surprisingly, virtually all cultured muscle cells express little or no GLUT4. We show here that adenovirus-mediated expression of the transcriptional coactivator PGC-1, which is expressed in muscle in vivo but is also deficient in cultured muscle cells, causes the total restoration of GLUT4 mRNA levels to those observed in vivo. This increased GLUT4 expression correlates with a 3-fold increase in glucose transport, although much of this protein is transported to the plasma membrane even in the absence of insulin. PGC-1 mediates this increased GLUT4 expression, in large part, by binding to and coactivating the muscle-selective transcription factor MEF2C. These data indicate that PGC-1 is a coactivator of MEF2C and can control the level of endogenous GLUT4 gene expression in muscle.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

The acute effects of contraction and insulin on the glucose transport and GLUT4 glucose transporter translocation were investigated in rat soleus muscles by using a 3-O-methylglucose transport assay and the sensitive exofacial labeling technique with the impermeant photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-y loxy)-2- propylamine (ATB-BMPA), respectively. Addition of wortmannin, which inhibits phosphatidylinositol 3-kinase, reduced insulin-stimulated glucose transport (8.8 +/- 0.5 mumol per ml per h vs. 1.4 +/- 0.1 mumol per ml per h) and GLUT4 translocation [2.79 +/- 0.20 pmol/g (wet muscle weight) vs. 0.49 +/- 0.05 pmol/g (wet muscle weight)]. In contrast, even at a high concentration (1 microM), wortmannin had no effect on contraction-mediated glucose uptake (4.4 +/- 0.1 mumol per ml per h vs. 4.1 +/- 0.2 mumol per ml per h) and GLUT4 cell surface content [1.75 +/- 0.16 pmol/g (wet muscle weight) vs. 1.52 +/- 0.16 pmol/g (wet muscle weight)]. Contraction-mediated translocation of the GLUT4 transporters to the cell surface was closely correlated with the glucose transport activity and could account fully for the increment in glucose uptake after contraction. The combined effects of contraction and maximal insulin stimulation were greater than either stimulation alone on glucose transport activity (11.5 +/- 0.4 mumol per ml per h vs. 5.6 +/- 0.2 mumol per ml per h and 9.0 +/- 0.2 mumol per ml per h) and on GLUT4 translocation [4.10 +/- 0.20 pmol/g (wet muscle weight) vs. 1.75 +/- 0.25 pmol/g (wet muscle weight) and 3.15 +/- 0.18 pmol/g (wet muscle weight)]. The results provide evidence that contraction stimulates translocation of GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.

High fat diet-induced hyperglycemia: prevention by low level expression of a glucose transporter (GLUT4) minigene in transgenic mice.

High-fat intake leading to obesity contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM, type 2). Similarly, mice fed a high-fat (safflower oil) diet develop defective glycemic control, hyperglycemia, and obesity. To assess the effect of a modest increase in the expression of GLUT4 (the insulin-responsive glucose transporter) on impaired glycemic control caused by fat feeding, transgenic mice harboring a GLUT4 minigene were fed a high-fat diet. Low-level tissue-specific (heart, skeletal muscle, and adipose tissue) expression of the GLUT4 minigene in transgenic mice prevented the impairment of glycemic control and accompanying hyperglycemia, but not obesity, caused by fat feeding. Thus, a small increase (< or = 2-fold) in the tissue level of GLUT4 prevents a primary symptom of the diabetic state in a mouse model, suggesting a possible target for intervention in the treatment of NIDDM.

Threshold effects of glucose transporter-4 (GLUT4) deficiency on cardiac glucose uptake and development of hypertrophy

The aim of this study was to investigate the metabolic and structural consequences of a decrease in glucose transporter-4 (GLUT4) levels on the heart. The CreLoxP system was utilised to delete GLUT4 in muscle tIssue including heart. The presence of the PGK-neoR cassette in the GLUT4-Lox mice resulted in reduced expression in all tIssues to levels 15-30% of wild-type control mice. In mice expressing Cre recombinase, there was a further reduction of GLUT4 in cardiac tIssue to almost undetectable levels. Cardiac glucose uptake was measured basally and during a uglycaemic/hyperinsulinaemic clamp using 2-deoxy-[1-(14)C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy. 2-deoxy-[1-14C]glucose. Insulin-stimulated glucose uptake was normal in hearts expressing 15% of normal GLUT4 levels but markedly reduced in mice with more profound reduction in GLUT4. Cardiac enlargement occurred only when GLUT4 levels were less than 5% of normal values. In heart there is a threshold level of GLUT4 above which insulin-stimulated glucose uptake is maintained. As little as 5% of normal GLUT4 levels expressed in heart is sufficient to prevent the development of cardiac hypertrophy.

AICAR administration affects glucose metabolism by upregulating the novel glucose transporter, GLUT8, in equine skeletal muscle

Equine metabolic syndrome is characterized by obesity and insulin resistance (IR). Currently, there is no effective pharmacological treatment for this insidious disease. Glucose uptake is mediated by a family of glucose transporters (GLUT), and is regulated by insulin-dependent and -independent pathways, including 5-AMP-activated protein kinase (AMPK). Importantly, the activation of AMPK, by 5-aminoimidazole- 4-carboxamide-1-D-ribofuranoside (AICAR) stimulates glucose uptake in both healthy and diabetic humans. However, whether AICAR promotes glucose uptake in horses has not been established. It is hypothesized that AICAR administration would enhance glucose transport in equine skeletal muscle through AMPK activation. In this study, the effect of an intravenous AICAR infusion on blood glucose and insulin concentrations, as well as on GLUT expression and AMPK activation in equine skeletal muscle (quantified by Western blotting) was examined. Upon administration, plasma AICAR rapidly reached peak concentration. Treatment with AICAR resulted in a decrease (P < 0.05) in blood glucose and an increase (P < 0.05) in insulin concentration without a change in lactate concentration. The ratio of phosphorylated to total AMPK was increased (P < 0.05) in skeletal muscle. While GLUT4 and GLUT1 protein expression remained unchanged, GLUT8 was increased (P < 0.05) following AICAR treatment. Up-regulation of GLUT8 protein expression by AICAR suggests that this novel GLUT isoform plays an important role in equine muscle glucose transport. In addition, the data suggest that AMPK activation enhances pancreatic insulin secretion. Collectively, the findings suggest that AICAR acutely promotes muscle glucose uptake in healthy horses and thus its therapeutic potential for managing IR requires investigation.

Expression of glucose transporter isoforms and the insulin receptor during hamster preimplantation embryo development

During preimplantation development, embryos of many species are known to express up to five isoforms of the facilitative glucose transporter proteins (GLUT). Development of hamster blastocysts is inhibited by glucose. We therefore investigated GLUT isoform and insulin receptor (IR) expression in hamster preimplantation embryos cultured in glucose-free medium from the 8-cell stage onwards. We show that GLUT1, 3 and 8 mRNA are constitutively expressed from the 8-cell to the blastocyst stage. The IR is expressed from the morula stage onwards. Messenger RNA of the insulin-responsive GLUT4 was not detected at any stage. GLUT1 and 3 were localised by immunocytochemistry. GLUT1 was expressed in both embryoblast and trophoblast, in the latter, mainly in basal and lateral membranes directed towards the blastocoel. and embryoblast. GLUT3 was exclusively localised in the apical. membrane of trophoblast cells. We show that hamster preimplantation embryos express several GLUT isoforms thus closely resembling embryos of other mammalian species. Despite endogenous IR expression, the insulin-sensitive isoform GLUT4 was not expressed, indicating that the insulin-mediated glucose uptake known from classical insulin target cells may not be relevant for hamster blastocysts.

Investigation of the Expression of Glucose Transporter Proteins in Human Cancer Cells

Cancer cells are known to display increased glucose uptake and consumption. The glucose transporter (GLUT) proteins facilitate glucose uptake, however, their exact role in cancer metabolism remains unclear. The present study examined mRNA and protein expression of GLUT1, GLUT3, GLUT4 and GLUT12 in lung, breast and prostate cancer cells and corresponding noncancerous cells. Additionally, GLUT expression was determined in tumours from mice xenografted with human cancer cells. Differences in the mRNA and protein expression of GLUTs were found between cancerous and corresponding noncancerous cells. These findings demonstrate abundant expression of GLUT1 in cancer and highlight the importance of GLUT3 as it was expressed in several cancer cells and tumours. GLUT expression patterns in vitro were supported by the in vivo findings. The study of GLUT protein expression in cancer is important for understanding cancer metabolism and may lead to identification of biomarkers of cancer progression and development of target therapies.

Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase

Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.

Glucose transporter expression in eutopic endometrial tissue and ectopic endometriotic lesions

Endometriosis is an extremely prevalent disorder characterized by the growth of endometrial tissue at ectopic locations. Glycolysis is an energy-producing mechanism that occurs in almost all cells and requires an adequate uptake of glucose mediated by glucose transporter (GLUT) proteins. At present, however, very little is known about their expression in either the endometrium or the endometriotic lesions. The objective of this study was to examine the expression of SLC2A genes in the endometrium of women with and without endometriosis and in the matching ectopic tissue, and to confirm the presence of the GLUT proteins in ectopic lesions. There was a significantly higher expression of SLC2A3 and a significantly lower expression of SLC2A4 in women with endometriosis compared with those without. In women with endometriosis, the ectopic expression of SLC2A3, SLC2A4 and SLC2A5 was significantly higher than that observed in the matching eutopic tissue. GLUT1 protein expression was present in both epithelial and stromal cells and GLUT3 was confined to CD45-positive leukocytes. GLUT4 expression was strong in both ectopic epithelial and stromal cells and localized to the cellular membrane in epithelial cells. These results show that GLUT expression is altered between eutopic and ectopic tissue and between women with and without endometriosis, and that GLUT4 may represent a significant entry route for glucose into the endometriotic epithelial cells. The inducible nature of GLUT4 and its limited cellular expression may make GLUT4 an attractive target for non-hormone-based treatments of endometriosis.

Development of an in vitro reconstitution assay for glucose transporter 4 translocation

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca2+-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5′-[γ-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein–protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes

Insulin and guanosine-5′-O-(3-thiotriphosphate) (GTPγS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (∼30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPγS response was significantly attenuated (∼85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPγS-stimulated GLUT4 translocation by ∼40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPγS-stimulated GLUT4 translocation but inhibited the insulin response by ∼40%. GTPγS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (∼50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPγS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473 - 484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.

Facilitative glucose transporter Glut1 is actively excluded from rod outer segments.

Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.