Surface GLUT4 Research Articles

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Naturally Overcomes Tumor Microenvironment (TME) Metabolic Challenges, Retaining Anti-Tumor Potency

Cellular engineering revolutionized adoptive cell therapy (ACT) through improved antigen recognition and cellular activation. Cancer cells are metabolically active and extensively consume nutrients from the TME, rendering it nutrient-deprived, acidic, and hypoxic, and resulting in intratumoral metabolic demands which negatively impact ACT antitumor activity. This is central to T cells, however, some of these challenges can be overcome through engineering, an ideal solution would be to have a cellular product that is inherently adept at overcoming these challenges. W-NK is a cytokine-reprogrammed, expanded, cryopreserved, ML-NK cell product derived from healthy human peripheral blood mononuclear cells; it comes with naturally effective potency towards tumor cell killing, and importantly can overcome metabolic challenges present in the TME. We aim at deciphering what metabolic features make W-NK particularly potent in adverse TME. W-NK characterization of metabolic fitness/adaptability was with transcriptomics (sc-RNA-seq; 10X Genomics) flow cytometry, bioenergetics (Seahorse Real-Time Cell Metabolic Analysis; Agilent), and proteomic (Sciex Zeno TOF 7600 tandem-mass spectrometry). The aforementioned assays evaluated the effect of differing media/TME on W-NK: 1) conventional (N) media (pH 7, glucose 11 mM); 2) TME-aligned media (pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1); and 3) ascites derived from patients with malignancy. Media composition was analyzed biochemically using clinical assays (Core Lab Clinical Studies at Washington University, St. Louis, USA), and by immune secretome profiling (Nomic Bio, Quebec, CA). At baseline, W-NK cells have a unique metabolic phenotype, with higher expression of cell surface nutrient transporters, e.g., GLUT1, CD98, ASCT2, MCT1 and PiT1, compared to conventional NK cells (cNK); additionally, W-NK metabolism was consistent with aerobic (“Warburg”) glycolysis, with ~80% of ATP coming from glycolysis vs. ~30% for cNK. In TME/ascites media, W-NK surface nutrient receptor expression adapted to meet metabolic demand. For example, transporters for amino acids, lactate and pyruvate are upregulated in these hypoglycemic medias. This coincided with a 50% shift in ATP manufacturing from glycolysis to mitochondria, suggesting that nutrients bypassed glycolysis to enter tricarboxylic acid cycle and oxidative phosphorylation. Proteomic analyses of W-NK cells cultured with either TME-aligned media or ascites supported this hypothesis, with up-regulation of metabolic pathways (ShinyGO) capturing amino-acid and lipid metabolism, and mitochondrial function. Overall, W-NK retained cytotoxic function in TME and ascites, as well as in native TME-aligned 3D assays from primary surgical tumor samples. Conversely, cNKs did not adapt to TME media, whether in nutrient transporter expression or ATP manufacturing, and demonstrated reduced survival and function, including cytotoxicity. W-NK has enhanced metabolic fitness, flexibility, and plasticity. This enables W-NK to utilize diverse macronutrients, engage different metabolic pathways, and maintain optimal ATP manufacturing. Overall, W-NK inherently survives and maintains function in the TME, a limiting factor for immune cell-based ACT. These data herald the promise of NK cell therapy; a Phase 1 clinical study of W-NK in acute myeloid leukemia is currently open and enrolling patients (NCT# 05470140).

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localization of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.

Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate.

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels.MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.

Effect of Taurine on the Regulation of Glucose Uptake in the Skeletal Muscle.

Diabetes is so common in Japan as to be called a national disease. Taurine, a free amino acid found abundantly in mammalian tissues that is also a key ingredient of many "energy drinks," has been shown to be effective in improving the hyperglycemic state caused by diabetes. Taurine administration is associated with increased insulin secretion from the pancreas, higher levels of insulin signaling-related factors, and higher expression of the glucose transporter, GLUT4. Skeletal muscle is the main target organ of insulin: Via cell surface GLUT4 molecules, myocytes take up blood glucose, enabling skeletal muscle contraction. The enhancing effect of taurine on blood glucose uptake in skeletal muscle has not been fully studied, and little is known about its mechanism. This review article summarizes what is known about the effects of taurine on insulin secretion from the pancreas and especially blood glucose uptake in skeletal muscle.

Knockout of syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion.

ABSTRACTAdipocytes 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.

Mitochondrial reactive oxygen species trigger metformin-dependent antitumor immunity via activation of Nrf2/mTORC1/p62 axis in tumor-infiltrating CD8T lymphocytes

BackgroundMetformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However,...

α-Synuclein Regulates Peripheral Insulin Secretion and Glucose Transport.

AimPopulation based studies indicate a positive association between type 2 diabetes (T2D) and Parkinson’s disease (PD) where there is an increased risk of developing PD in patients with T2D. PD is characterized by the abnormal accumulation of intraneuronal aggregated α-synuclein (α-syn) in Lewy bodies, which negatively impact neuronal viability. α-syn is also expressed in both pancreatic islets and skeletal muscle, key players in glucose regulation. Therefore, we examined the functional role of α-syn in these tissues.MethodsUsing mice lacking, overexpressing or transiently injected with α-syn, effects on glucose and insulin tolerance and insulin secretion were determined, with further characterization of the effects on GLUT4 translocation using GLUT4myc myotubes.ResultsMice genetically ablated for α-syn became glucose intolerant and insulin resistant with hyperinsulinemia and reduced glucose-stimulated insulin secretion (GSIS). Mice overexpressing human α-syn are more insulin senstive and glucose tolerant compared to controls with increased GSIS. Injection of purified α-syn monomers also led to improved glucose tolerance and insulin sensitivity with hightened GSIS. α-syn monomer treatments increased surface GLUT4 levels in myotubes but without any significant change in Akt phosphorylation. The increase in cell surface GLUT4 was largely due to a large reduction in GLUT4 endocytosis, however, with a compensatory reduction in GLUT4 exocytosis.ConclusionCumulatively, this data suggests that α-syn modulates both pancreatic beta cell function and glucose transport in peripheral tissues, thereby playing a pivitol role in the maintenance of normal glucose homeostasis.

CD8 co-receptor links T cell avidity and metabolism

Abstract This study investigated the mechanistic basis behind the reported superior efficacy of high-avidity CD8 T cells. CMV and HIV are both chronic viral infections, but while CMV-specific CD8 T cells can mediate lifelong viral control, in untreated HIV infection HIV-specific CD8 T cells progressively lose function. Using in vitro studies of human cells, we show that avidity-dependent downregulation of the CD8 co-receptor directly programs metabolism, due to a novel association between CD8 and the glucose transporter GLUT1. We used flow cytometry to profile ex vivo and virus-specific CD8 T cells from HIV-infected individuals on antiretroviral therapy. Ex vivo, cells expressing low levels of CD8 (CD8dim) expressed more CD69 but less cell surface GLUT1, and took up less glucose (2-NBDG) than CD8bright T cells. Following antigen stimulation, CD3, CD8, and GLUT1 were downregulated from the cell surface in an avidity-dependent manner. CMV-specific CD8 T cells, which were of higher avidity, downregulated these proteins to a greater extent than lower-avidity HIV-specific CD8 T cells. GLUT1 downregulation strongly correlated with CD8 but not CD3 downregulation. Antibody-mediated downregulation of CD8 from the cell surface resulted in reduced glucose uptake and increased fatty acid (Bodipy) uptake, independent of CD3. Finally, CD3, CD8, and GLUT1 downregulation by HIV-specific CD8 T cells was impaired following viral escape mutations that reduced CD8 T cell avidity. We confirmed this finding in a transduction setting with a single clonal TCR. Our data reveal a novel function of the CD8 co-receptor, linking the avidity and metabolism of CD8 T cells.

Abstract 15747: The Sarcoplasmic Reticulum Calcium ATPase Pump Modulates Whole-body Glucose Homeostasis Through a Protein Disulfide Isomerase-dependent Pathway

Introduction: The heart is a very active metabolic organ which exerts endocrine effects by secreting cardiokines. However, its role in regulating whole-body metabolism has received scarce attention. Hypothesis: We hypothesized that cardiac-specific overexpression of the sarcoplasmic reticulum calcium ATPase (SERCA) pump, which tightly regulates cytosolic calcium, modulates whole-body glucose homeostasis by secreting cardiokines during healthy and diabetic states. Methods: Diabetes was induced by streptozotocin in wild type (WT) and transgenic (TG) mice overexpressing the SERCA pump in the heart (n=6-10/group). Translocation of glucose transporters (GLUTs) to the cell surface was measured via a photolabeling biotinylation assay. Glucose and palmitate oxidation rates, cardiac work and ATP production were quantified in isolated working hearts. Results: Blood glucose levels were lower in diabetic TG mice compared to diabetic WT mice (P=0.023). Diabetes induced a downregulation of cell surface GLUT4 protein content in cardiac and skeletal muscle (by 56% and 79%, respectively, P<0.05), as well as in white and brown adipose tissue of WT mice (by 68% and 74%, respectively, P<0.05), which was rescued in TG diabetic mice. WT diabetic hearts had decreased cardiac glucose oxidation rates and cardiac efficiency, and increased palmitate oxidation rates (P<0.05), which were restored in TG diabetic hearts. Using quantitative mass spectrometry, we found protein disulfide isomerase (PDI) upregulated in the heart of TG mice (by 1822%, P<0.0001), which was confirmed in the serum of TG mice via an ELISA assay (by 243%, P=0.03). Inoculation with exogenous PDI reduced blood glucose in WT healthy mice and partially rescued hyperglycemia in WT diabetic mice compared to untreated counterparts (P=0.009 and P=0.044, respectively). Conclusions: These data suggest that cardiac-specific SERCA overexpression rescues hyperglycemia by improving glucose transport in striated muscle and adipose tissue of diabetic TG mice. Identification of the pathways by which the SERCA pump and cardiac secreted proteins (i.e. PDI) are involved in the regulation of whole-body glucose homeostasis could lead to the identification of novel pharmacological targets in diabetic patients.

TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes

Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.

Knockdown of long noncoding RNA dreh facilitates cell surface GLUT4 expression and glucose uptake through the involvement of vimentin in 3T3-L1 adipocytes

Glucose uptake in adipocytes is crucial for regulating systemic metabolism. Long noncoding RNAs (lncRNAs), defined as being transcripts with lengths exceeding 200 nucleotides that are not translated, are recently identified regulators of cellular functions. Previously, we have shown that an lncRNA, “down-regulated expression by hepatitis B virus X” (dreh), is involved in glucose transport in skeletal muscle cells. Here, we aimed to examine the involvement of dreh in glucose transport in 3T3-L1 adipocytes. Expression analysis showed that dreh was expressed in 3T3-L1 fibroblasts and adipocytes. Knockdown of dreh expression using its specific siRNAs lowered the glucose concentration of the medium and facilitated [3H]-2-deoxyglucose transport in adipocytes. Additionally, dreh silencing enhanced the protein expression of glucose transporter (GLUT4) in the plasma membrane of adipocytes. Treatment with siRNA against vimentin attenuated the glucose-lowering effect of dreh depletion. These results suggest that the repression of dreh facilitates glucose transport via increased GLUT4 expression in the plasma membrane through the involvement of vimentin in 3T3-L1 adipocytes. In conclusion, dreh is the first observed lncRNA that regulates glucose transport in adipocytes and could serve as a novel therapeutic target for diabetes by modulating adipocyte function. Considering the new function of dreh, we propose that dreh be renamed “down-regulated expression-related hexose/glucose transport enhancer.”

Microrna-31 Regulates T-Cell Metabolism Via HIF1α and Promotes Effector Function

MicroRNAs (miRs) repress gene expression at the post-transcriptional level via binding target mRNAs subsequently promoting mRNA degradation or impeding translation. Certain miRs influence the survival, differentiation, and function of T cells in cancer, infections, and autoimmunity. Uncontrolled T-cell allogeneic responses contribute to chronic graft-versus-host disease (cGVHD), a major cause of non-relapse mortality following allogeneic hematopoietic stem-cell transplantation (allo-HCT). Dysregulation of miR-31 is implicated in cancer, but how miR-31 impacts allogeneic T-cell response is unclear. Using oligonucleotide array, we found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in recipient mice following bone marrow transplantation (BMT) (Figure 1A-B). To understand how miR-31 regulates T-cell responses to alloantigens, we utilized murine models of allogeneic BMT and donor mice with miR-31 conditional knock-out on their T cells. In a sclerodermatous cGVHD model (B6→BDF1), we observed that the recipients transferred with donor T cells deficient for miR-31 exhibited significantly alleviated disease (Figure 1C), reflected by attenuated fibrosis and pathologic damage in skin. In a bronchiolitis obliterans cGVHD model (B6→B10.BR), we found that the recipients of miR-31-deficient T cells had reduced airway resistance, elastance (Figure 1D) and pathological damage in the lungs as compared with those of WT T cells. The role of miR-31 in promoting T-cell pathogenicity was further confirmed when miR-31 was inhibited by administration of specific antagomir (locked nuclear acid anti-miR-31). We used mass cytometry to analyze donor immune cell reconstitution in recipient spleens after BMT. Consistent with attenuated disease manifestation, the recipients of miR-31-deficient T cells displayed improved reconstitution of donor T- and B- lymphocytes. The follicular T helper cells (TFH) instruct germinal center (GC) B-cell expansion, affinity maturation, and plasma cell differentiation, whereas follicular regulatory T cells (TFR) inhibit TFH -mediated B-cell activation and antibody production. We found that miR-31 deficient T cells differentiated into more TFR, but fewer TFH during alloresponses. As a result, the differentiation and activation of GC B cells and the generation of plasma cells were attenuated in the recipients of miR-31-deficient T cells. Furthermore, miR-31-deficient T cells exhibited defects in proliferation and survival in allogeneic recipients, resulting in fewer donor T cells in recipient thymus, skin and lungs. In the absence of miR-31, T cells differentiated less into Th17 cells but more towards Tregs (Figure 1E) in vivo. Those miR-31-deficient Tregs expressed higher levels of Neuropilin 1 and PD-L1, the markers associated with superior stability and suppressive function of Tregs. While miR-31 played little role on Th1 cell differentiation, it facilitated alloantigen-reactive iTregs losing Foxp3 and producing IFNγ after being transferred into allogeneic recipients. Hypoxia, a hallmark of inflamed and damaged tissue, can drive fibrosis and disease development through immune cell dysregulation. Upon activation, T cells rapidly increase their metabolic rate and switch from oxidative phosphorylation to aerobic glycolysis. We found that miR-31-deficient T cells exhibited reduced hypoxia-inducible factor 1α (HIF1α) signaling (glycolysis promotor), surface GLUT1 expression, and glucose uptake, but increased lipid droplet accumulation in allogeneic recipients, suggesting that miR-31 promotes metabolic switch from fatty acid β-oxidation to aerobic glycolysis in allo-reactive T cells (Figure 1F). Furthermore, under hypoxia (3% oxygen) but not normoxia (21% oxygen) conditions, miR-31 increased Th17 but decreased iTreg differentiation from naïve CD4 T cells in the presence of IL-6 and TGFβ in vitro. Taken together, miR-31 regulates T-cell expansion, differentiation, and metabolism via promoting HIF1α expression and hypoxia adaptation in allo-reactive T cells, which enhances T-cell effector function and pathogenicity after allogeneic BMT. Disclosures No relevant conflicts of interest to declare.

The Sarcoplasmic Reticulum Calcium ATPase Pump Is a Major Regulator of Whole–Body Glucose Metabolism

The heart is a very active metabolic organ which exerts endocrine effects by secreting natriuretic peptides. However, its role in the regulation of whole-body metabolism has received scarce attention. Therefore, we hypothesized that cardiac-specific overexpression of the sarcoplasmic reticulum calcium ATPase (SERCA) pump, which tightly regulates cytosolic calcium, modulates whole-body glucose homeostasis during healthy and diabetic states. Diabetes was induced by streptozotocin injections in wild type (WT) and transgenic (TG) mice overexpressing the SERCA pump in the heart (n=4-10/group). Translocation of glucose transporters (GLUTs) was measured in insulin-sensitive tissues using a photolabeled biotinylation assay. The cardiac secretome function was analyzed by mass spectrometry. We reported that blood glucose concentration was lower in diabetic TG mice compared to diabetic WT mice (p=0.023). Cell surface GLUT-4 and -8 protein content was downregulated in the heart of WT diabetic vs. healthy mice (by 56% and 71%, p=0.01 and p<0.001, respectively), which was rescued in TG diabetic mice. In addition, diabetes induced a downregulation of cell surface GLUT4 trafficking in skeletal muscle, and white and brown adipose tissue of WT mice (by 79%, 68% and 74%, respectively, P<0.05), which was restored in TG diabetic mice. We further identified 28 and 32 candidate proteins that have a high probability (i.e., > 95%) of being secreted by the ventricle and atria of TG mice, respectively. Of these, 13 and 10 potentially secreted proteins by the atria and ventricle, respectively, were involved in metabolic processes. In conclusion, these data suggest that cardiac-specific SERCA overexpression rescued hyperglycemia by improving glucose transport in insulin-sensitive tissues of diabetic TG mice. Insights gained from this study could identify the SERCA pump and its secreted proteins as novel pharmacological targets to regulate glucose homeostasis in diabetic patients. Disclosure Z. Maria: None. A. Campolo: None. S. Shoop Gray: None. S. Hartson: None. V. Lacombe: None.

AMPK/AS160 mediates tiliroside derivatives-stimulated GLUT4 translocation in muscle cells.

IntroductionThe Chinese herb Potentilla chinensis can reduce blood glucose level of diabetic mice. Tiliroside is the main effective component, but the detailed mechanism is not clear. Skeletal muscles play an important role in whole body glucose homeostasis. Insulin and exercise/contraction stimulate glucose uptake by muscle cells via redistribution of glucose transporter GLUT4 to the cell surface.Materials and methodsWe explored the effects of tiliroside derivatives on cell surface GLUT4 level (GLUT4 translocation) and the underlying mechanism in L6-GLUT4myc muscle cells.ResultsWe showed that tiliroside derivatives D1–22 stimulated GLUT4myc translocation in L6-GLUT4myc skeletal muscle cells. Derivatives D1, D8 and D18 regulated GLUT4myc translocation in a time- and dose-dependent manner. Their effects on GLUT4 were additive with that of acute insulin stimulation. Moreover, they increased phosphorylated adenosine monophosphate-activated protein kinase (AMPK), but not protein kinase B (PKB, also called Akt). Their effects on GLUT4 were inhibited by Compound C. In addition, derivative D8 significantly stimulated AMPK and Akt substrate of 160 kDa (AS160) phosphorylation and GLUT4myc translocation in L6-GLUT4myc cells, but not in L6-AS160 4A-GLUT4myc cells.ConclusionTiliroside derivatives D1, D8 and D18 stimulated GLUT4myc translocation by a mechanism different to that of insulin in skeletal muscle cells. The effect of derivative D8 on GLUT4myc translocation is mediated by AMPK/AS160 signaling pathway.

Quercetin inhibits glucose transport by binding to an exofacial site on GLUT1

Quercetin, a common dietary flavone, is a competitive inhibitor of glucose uptake and is also thought to be transported into cells by GLUT1. In this study, we confirm that quercetin is a competitive inhibitor of GLUT1 and also demonstrate that newly synthesized compounds, WZB-117 and BAY-876 are robust inhibitors of GLUT1 in L929 cells. To measure quercetin interaction with L929 cells, we develop a new fluorescent assay using flow cytometry. The binding of quercetin and its inhibitory effects on 2-deoxyglucose (2DG) uptake showed nearly identical dose dependent effects, with both having maximum effects between 50 and 100 μM and similar half maximum effects at 8.9 and 8.5 μM respectively. The interaction of quercetin was rapid with t1/2 of 54 s and the onset and loss of its inhibitory effects on 2DG uptake were equally fast. This suggests that either quercetin is simply binding to surface GLUT1 or its transport in and out of the cell reaches equilibrium very quickly. If quercetin is transported, the co-incubation of quercetin with other glucose inhibitors should block quercetin uptake. However, we observed that WZB-117, an exofacial binding inhibitor of GLUT1 reduced quercetin interaction, while cytochalasin B, an endofacial binding inhibitor, enhanced quercetin interaction, and BAY-876 had no effect on quercetin interaction. Taken together, these data are more consistent with quercetin simply binding to GLUT1, but not actually being transported into L929 cells via the glucose channel in GLUT1.

Dioleoylphosphoethanolamine Retains Cell Surface GLUT4 by Inhibiting PKCα-Driven Internalization

Background/Aims: Phosphatidylethanolamine, a component of the plasma membrane, regulates diverse cellular processes. The present study investigated the role of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the trafficking of the glucose transporter GLUT4 and the glucose homeostasis. Methods: Monitoring of GLUT4 trafficking, GLUT4 internalization assay, and glucose uptake assay were carried out using differentiated 3T3-L1-GLUT4myc adipocytes. Akt1/2 and PKC isozymes were knocked-down by transfecting each siRNA. Cell-free PKC assay and in situ PKCα assay with a FRET probe were carried out. Oral glucose tolerance test (OGTT) was performed using BKS.Cg-+Lep^db/+Leb^db/Jcl mice, an animal model of type 2 diabetes mellitus (DM). Results: DOPE increased cell surface localization of the glucose transporter GLUT4 in differentiated 3T3-L1-GLUT4myc adipocytes, regardless of Akt activation. Likewise, PKCα deficiency increased cell surface localization of GLUT4, that occludes the effect of DOPE. DOPE clearly suppressed phorbol 12-myristate 13-acetate-induced PKCα activation in the cell-free and in situ PKC assay. DOPE and PKCα deficiency cancelled endocytic internalization of GLUT4 localized on the plasma membrane after insulin stimulation. DOPE significantly enhanced glucose uptake into cells. A similar effect was obtained by knocking-down PKCα, that occludes the effect of DOPE. In OGTT, oral administration with DOPE effectively restricted an increase in the blood glucose levels after glucose loading in type 2 DM model mice. Conclusion: The results of the present study show that DOPE retains cell surface GLUT4 by suppressing PKCα-driven endocytic internalization of GLUT4, to enhance glucose uptake into cells and restrict an increase in the blood glucose levels after glucose loading in type 2 DM.

Electrical pulse stimulation induces GLUT4 translocation in C2C12 myotubes that depends on Rab8A, Rab13, and Rab14.

The signals mobilizing GLUT4 to the plasma membrane in response to muscle contraction are less known than those elicited by insulin. This disparity is undoubtedly due to lack of suitable in vitro models to study skeletal muscle contraction. We generated C2C12 myotubes stably expressing HA-tagged GLUT4 (C2C12-GLUT4 HA) that contract in response to electrical pulse stimulation (EPS) and investigated molecular mechanisms regulating GLUT4 HA. EPS (60 min, 20 V, 1 Hz, 24-ms pulses at 976-ms intervals) elicited a gain in surface GLUT4 HA (GLUT4 translocation) comparably to insulin or 5-amino imidazole-4-carboxamide ribonucleotide (AICAR). A myosin II inhibitor prevented EPS-stimulated myotube contraction and reduced surface GLUT4 by 56%. EPS stimulated AMPK and CaMKII phosphorylation, and EPS-stimulated GLUT4 translocation was reduced in part by small interfering (si)RNA-mediated AMPKα1/α2 knockdown, compound C, siRNA-mediated Ca2+/calmodulin-dependent protein kinase (CaMKII)δ knockdown, or CaMKII inhibitor KN93. Key regulatory residues on the Rab-GAPs AS160 and TBC1D1 were phosphorylated in response to EPS. Stable expression of an activated form of the Rab-GAP AS160 (AS160-4A) diminished EPS- and insulin-stimulated GLUT4 translocation, suggesting regulation of GLUT4 vesicle traffic by Rab GTPases. Knockdown of each Rab8a, Rab13, or Rab14 reduced, in part, GLUT4 translocation induced by EPS, whereas only Rab8a, or Rab14 knockdown reduced the AICAR response. In conclusion, EPS involves Rab8a, Rab13, and Rab14 to elicit GLUT4 translocation but not Rab10; moreover, Rab10 and Rab13 are not engaged by AMPK activation alone. C2C12-GLUT4 HA cultures constitute a valuable in vitro model to investigate molecular mechanisms of contraction-stimulated GLUT4 translocation.

ID:3002 Notch-mediated control of memory T cells against cancer cells

T cells recognize an antigen presented by self-MHC, and the part of initially activated T cells differentiate toward memory T cells. T cells also recognize cancer cells leading to generation of memory T cells against cancer-derived antigens although the activity of T cells are frequently suppressed by various factors. The release from T cell inhibitory factors could allow T cells to respond to cancer cells. However, it remains unclear which molecules are required for long-term survival of memory T cells and generation of memory T cells against cancer cells. Notch functions as a regulator for fate decision, activation and survival of immune cells. We have demonstrated the roles of Notch in mature T cell differentiation and found that Notch signaling is essential for the maintenance of memory CD4 T cells. The inhibition of Notch disturbs the survival of memory CD4 T cells. The effect of Notch on T cell survival depended on glucose uptake through cell surface Glut1 expression. We revealed that Notch is crucial for the long-term survival of memory T cells against cancer cells and suppression of Notch signaling reduced the tumor antigen-specific killing of cancer cells. Those data demonstrate that Notch is pivotal for the maintenance of memory T cells against cancer cells and suggest that activation of Notch signaling might be advantageous to cancer immunotherapy.

Effect of Lithium on the Mechanism of Glucose Transport in Skeletal Muscles.

While lithium is known to stimulate glucose transport into skeletal muscle, the mechanisms of the increased glucose transport by lithium in skeletal muscle are not well defined yet. We excised epitrochlearis muscles from male Wistar rats and measured the transport rates of a glucose analog into lithium-, insulin-, and muscular contraction-stimulated skeletal muscle tissue and we also analyzed the levels of cell surface glucose transport 4 using a photolabeling and multicolor immunofluorescence method. In addition, we generated a cell line that stably expresses myc-tagged GLUT4 to measure the rates of GLUT4 internalization and externalization. Lithium significantly increased 2-DG glucose transport rate in skeletal muscles; however, it was significantly lower than the stimulation induced by the maximum concentration of insulin or tetanic contraction. But co-treatment of lithium with insulin or tetanic contraction increased glucose transport rate by ∼200% more than lithium alone. When skeletal muscle tissues were treated with lithium, insulin, and muscular contraction, the levels of cell surface GLUT4 protein contents were increased similarly by ∼6-fold compared with the basal levels. When insulin or lithium stimuli were maintained, the rate of GLUT4myc internalization was significantly lower, and lithium was found to suppress the internalization of GLUT4myc more strongly. The lithium-induced increase in glucose uptake of skeletal muscles appears to increase in cell surface GLUT4 levels caused by decreased internalization of GLUT4. It is concluded that co-treatment of lithium with insulin and muscular contraction had a synergistic effect on glucose transport rate in skeletal muscle.