Human GLUT1 Research Articles

The role of salt bridge networks in the stability of the yeast hexose transporter 1

BackgroundThe yeast S. cerevisiae preferably metabolizes glucose through aerobic glycolysis. Glucose transport is facilitated by multiple hexose transporters (Hxts), and their expression and activity are tightly regulated by multiple mechanisms. However, detailed structural and functional analyses of Hxts remain limited, largely due to the lack of crystal structure. MethodsHomology modeling was used to build a 3D structural model for the yeast glucose transporter Hxt1 and investigate the effects of site directed mutations on Hxt1 stability and glucose transport activity. ResultsThe conserved salt bridge-forming residues observed in the human Glut4 and the yeast glucose receptor Rgt2 were identified within and between the two 6-transmembrane spanning segments of Hxt1. Most of the RGT2 mutations that disrupt the salt bridge networks were known to cause constitutive signal generation, whereas the corresponding substitutions in HXT1 were shown to decrease Hxt1 stability. While substitutions of the two residues in the salt bridge 2 in Glut4—E329Q and E393D—were reported to abolish glucose transport, the equivalent substitutions in Hxt1 (D382Q and E454D) did not affect Hxt1 glucose transport activity. ConclusionsSubstitutions of equivalent salt bridge-forming residues in Hxt1, Rgt2, and Glut4 are predicted to lock them in an inward-facing conformation but lead to different functional consequences. General significanceThe salt bridge networks in yeast and human glucose transporters and yeast glucose receptors may play different roles in maintaining their structural and functional integrity.

The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL.

Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding.

Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin

Glucose transporters are gatekeepers of cellular glucose metabolism. Understanding how their activity is regulated can provide insight into mechanisms of glucose homeostasis and diseases arising from dysregulation of glucose transport. Glucose stimulates endocytosis of the human glucose transporter GLUT1, but several important questions remain surrounding the intracellular trafficking itinerary of GLUT1. Here, we report that increased glucose availability triggers lysosomal trafficking of GLUT1 in HeLa cells, with a subpopulation of GLUT1 routed through ESCRT-associated late endosomes. This itinerary requires the arrestin-like protein TXNIP, which interacts with both clathrin and E3 ubiquitin ligases to promote GLUT1 lysosomal trafficking. We also find that glucose stimulates GLUT1 ubiquitylation, which promotes its lysosomal trafficking. Our results suggest that excess glucose first triggers TXNIP-mediated endocytosis of GLUT1 and, subsequently, ubiquitylation to promote lysosomal trafficking. Our findings underscore how complex coordination of multiple regulators is required for fine-tuning of GLUT1 stability at the cell surface.

Identification of Structural Determinants of the Transport of the Dehydroascorbic Acid Mediated by Glucose Transport GLUT1

GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1's transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the Vmax compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C's transport via GLUT1.

Genetic Modulation of the GLUT1 Transporter Expression-Potential Relevance in Complex Diseases.

The human GLUT1 (SLC2A1) membrane protein is the key glucose transporter in numerous cell types, including red cells, kidney, and blood-brain barrier cells. The expression level of this protein has a role in several diseases, including cancer and Alzheimer's disease. In this work, to investigate a potential genetic modulation of the GLUT1 expression level, the protein level was measured in red cell membranes by flow cytometry, and the genetic background was analyzed by qPCR and luciferase assays. We found significant associations between red cell GLUT1 levels and four single nucleotide polymorphisms (SNP) in the coding SLC2A1 gene, that in individuals with the minor alleles of rs841848, rs1385129, and rs11537641 had increased, while those having the variant rs841847 had decreased erythrocyte GLUT1 levels. In the luciferase reporter studies performed in HEK-293T and HepG2 cells, a similar SNP-dependent modulation was observed, and lower glucose, serum, and hypoxic condition had variable, cell- and SNP-specific effects on luciferase expression. These results should contribute to a more detailed understanding of the genetic background of membrane GLUT1 expression and its potential role in associated diseases.

Cryo-EM structure of human glucose transporter GLUT4

GLUT4 is the primary glucose transporter in adipose and skeletal muscle tissues. Its cellular trafficking is regulated by insulin signaling. Failed or reduced plasma membrane localization of GLUT4 is associated with diabetes. Here, we report the cryo-EM structures of human GLUT4 bound to a small molecule inhibitor cytochalasin B (CCB) at resolutions of 3.3 Å in both detergent micelles and lipid nanodiscs. CCB-bound GLUT4 exhibits an inward-open conformation. Despite the nearly identical conformation of the transmembrane domain to GLUT1, the cryo-EM structure reveals an extracellular glycosylation site and an intracellular helix that is invisible in the crystal structure of GLUT1. The structural study presented here lays the foundation for further mechanistic investigation of the modulation of GLUT4 trafficking. Our methods for cryo-EM analysis of GLUT4 will also facilitate structural determination of many other small size solute carriers.

Glucose-assisted trophic conversion of Chlamydomonas reinhardtii by expression of glucose transporter GLUT1

The use of genetic engineering techniques to alter the nutritional patterns of microalgae is a key means of enhancing microalgal biomass, as it enables the highly efficient heterotrophic growth of microalgae via the utilisation of organic carbon sources, especially glucose. To investigate the effects of the expression of the glucose transporter on the growth of the model alga, Chlamydomonas reinhardtii, we transformed C. reinhardtii using the human GLUT1 gene, regulated by the Hsp70, PsaD, and Arg7 promoters, and obtained engineered algae after screening. The engineered algae showed better growth than the control algae under both heterotrophic and mixotrophic growth conditions. The engineered alga in which the GLUT1 gene was regulated by the Arg7 promoter exhibited the best growth. Although the engineered algae exhibited heterotrophic growth in complete darkness by using glucose as the carbon source, their growth rates and biomass were relatively low compared to those of the control. Analysis of the glucose transport ability of the engineered algae revealed that glucose was successfully transported to the cytoplasm via the glucose transporters, but was unable to enter the chloroplasts, which could explain the slow heterotrophic growth of the engineered algae.

Abstract 10743: Overexpression of GLUT4 Induces Pulmonary Hypertension

Introduction: Pulmonary arterial hypertension (PAH) induces significantly elevated glucose uptake in the patient lungs and right ventricles (RV), which correlates with the severity of the disease. However, the mechanisms responsible for the increased glucose uptake and its contribution to the PAH pathobiology remain unclear. We discovered that Sugen/hypoxia-induced PAH is associated with a marked overexpression of glucose transporter, GLUT4, starting from the early stage of the disease. Hypothesis: We investigated the role of GLUT4 overexpression in triggering PAH and evaluated the downstream mechanisms responsible for the PAH phenotype. Methods: In this study, we used the mouse model overexpressing human GLUT4 (hGLUT4). RV hemodynamics and function were assessed in hGLUT4 mice treated or not with IMPA1 inhibitor (L690,330, i.p., 22 mg/kg BW, daily for 6 weeks). Isolated pulmonary artery smooth muscle cells (PASMC) were used to perform glucose uptake assays and bioenergetics measurements. Results: hGLUT4 mice showed significantly increased RV systolic pressure (RVSP, 28.98±0.72, p<0.001, N=7-9) and RV hypertrophy (0.319±0.006, p<0.0001, N=7-9) compared to WTs and non-transgenic controls. GLUT4 overexpression significantly increased the glucose uptake in PASMC (~30% increase to control, p<0.01) and induced severe mitochondrial dysfunction in unstimulated and insulin-stimulated cells. Previously, we discovered that an increase in glucose uptake elevates the levels of glucose 6-phosphate (G6P) and activates the G6P metabolizing enzyme, myo-inositol monophosphatase 1 (IMPA1). We confirmed the increased expression of IMPA1, a critical activator of the inositol synthesis and inositol-dependent proliferative pathways in hGLUT4 mice. Notably, treatment with the IMPA1 inhibitor completely reversed PAH by reducing RVSP and RV hypertrophy. Conclusions: We conclude that increased GLUT4 expression is sufficient to induce spontaneous PAH through increased glucose uptake, initiation of mitochondrial dysfunction, and upregulation of IMPA1. This discovery introduces a novel potential target, IMPA1, that connects dysregulated glucose metabolism with activation of the proliferative signaling.

In-silico analysis of phylogenetic relationship and potentially damaging nsSNPs in human SLC2A2 gene

Transport of glucose across the eukaryotic cell membranes is carried out by members of the glucose transporter (GLUT) family which is mainly divided into three classes (I, II and III) on the basis of phylogenetic relationship. In humans, one member of Class I called GLUT2 is encoded by solute carrier family 2, facilitated glucose transporter member 2 (SLC2A2) gene located on third chromosome. Protein mediatedglucosemovement acrosscell membranes is made possible through GLUT2 that is atrans-membrane carrierprotein. It regulates the entry of glucose and secretion of insulin in the pancreatic cell. It has three isoforms and the longest isoform consists of 524 amino acid. There are 13 extracellular, 12 transmembrane and 5 cytoplasmic domains in human GLUT2. The risk of Fanconi-Bickel syndrome (FBS), diabetes, breast cancer (BC) and Alzheimer disease (AD) is associated with improper functioning of GLUT2. The most frequent form of genetic changes is single nucleotide polymorphism (SNPs). Non synonymous SNPs (nsSNPs) can result in alterations of amino acids and subsequent changes in phenotype. In this study, in-silico analysis was done to find phylogenetic relationship of human GLUT2 protein and possible deleterious effect of nsSNPs of its coding region. Clustal Omega was used to make phylogenetic tree. SIFT, PolyPhen, PROVEAN and SNPeffect were used to predict deleterious or tolerated SNPs. 167 nsSNPs were predicted to be damaging by SIFT, 65 to be possibly damaging and 77 to be probably damaging by PolyPhen. PROVEAN predicted 162 nsSNPs to be neutral and 138 to be deleterious. 101 SNPs were found to be damaging by three algorithms; SIFT, PolyPhen and PROVEAN.

Markers of Prognosis for Early Stage Cervical Cancer Patients (Stage IB1, IB2) Undergoing Surgical Treatment.

BackgroundFor individuals with cervical cancer, large tumor volume, lymph node metastasis, distant metastasis, and parauterine infiltration are usually associated with a poor prognosis. Individuals with stage 1B1 and 1B2 cervical cancer usually do not have these unfavorable prognostic factors. Once the disease progresses, the prognosis becomes extremely poor. Therefore, investigating the prognostic markers of these cervical cancer patients is necessary for treatment.MethodsThis retrospective study included 95 cervical cancer patients treated with surgery. The patients were divided into progressor and non-progressor groups according to postoperative follow-up results. T-test (or Mann−Whitney U test), chi-squared test (or Fisher’s exact test) and receiver operating characteristic (ROC) curves were used to evaluate imaging, hematology, and clinicopathological index differences between the two groups. Cox analysis was performed to select the independent markers of progression-free survival (PFS) when developing the nomogram. Validation of the nomogram was performed with 1000 bootstrapped samples. The performance of the nomogram was validated with ROC curves, generated calibration curves, and Kaplan-Meier and decision curve analysis (DCA).ResultsCervical stromal invasion depth, lymphovascular space invasion (LVSI), human papilloma virus (HPV-16), Glut1, D-dimer, SUVmax and SUVpeak showed significant differences between the two groups. Multivariate Cox proportional hazard model showed SUVpeak (p = 0.012), and HPV-16 (p = 0.007) were independent risk factors and were used to develop the nomogram for predicting PFS. The ROC curves, Kaplan-Meier method, calibration curves and DCA indicated satisfactory accuracy, agreement, and clinical usefulness, respectively.ConclusionsSUVpeak level (≥7.63 g/cm3) and HPV-16 negative status before surgery were associated with worse PFS for patients with cervical cancer. Based on this result, we constructed the nomogram and showed satisfactory performance. Clinically, individualized clinical decision-making can be performed on patients based on this result.

I dentification of a novel small‐molecule allosteric inhibitor of glucose transporter type 5 for treating fructose‐induced diseases

Glucose transporter type 5 (GLUT5) is a major molecule that transports fructose in mammals. Fructose has become a major constituent of our modern diet and been implicated as an underlying cause of metabolic diseases such as diabetes, obesity, and nonalcoholic fatty liver disease. In addition, certain cancer cells have been found to utilize fructose via the GLUT5-dependent pathway for their survival. Moreover, mutations in the ALDOB gene cause hereditary fructose intolerance, a condition in which individuals experience hypoglycemia upon fructose intake. These results suggest that limiting fructose utilization by inhibiting GLUT5 may be a novel therapeutic strategy for treating fructose-induced diseases. In this study, we report the identification of a potent, selective, and orally available small-molecule inhibitor of GLUT5. Through high-throughput screening and subsequent compound optimization, we identified compound S-700. S-700 exhibited a potent inhibitory effect on fructose uptake in human GLUT5- and mouse GLUT5-overexpressed Chinese hamster ovary cells with IC50 values of 59 and 28 nM, respectively. The in vitro analysis showed that S-700 allosterically binds to GLUT5 independent of the fructose binding site. In contrast, S-700 did not inhibit human GLUT1; GLUT1 inhibition should be avoided as it impairs the brain function. Single oral administration of S-700 (0.03–1 mg/kg) potently inhibited GLUT5-mediated fructose uptake in the gut during an oral fructose tolerance test in rats. Notably, the predose of S-700 (1 mg/kg) up to 24 h before a fructose dose was highly effective in inducing this effect, indicating the durable effect of S-700. We further assessed the effects of chronic oral administration of S-700 (0.1–3 mg/kg/day) on liver triglyceride (TG) accumulation in Western Diet (D12079B)-fed rats. S-700 (0.3–3 mg/kg) potently suppressed TG levels in the liver and improved the abnormal liver weight of these animals. The therapeutic potential of S-700 (0.3–3 mg/kg/day) in decreasing liver TG levels was similar to or more potent than that of PF-06835919 (30 mg/kg/day), a ketohexokinase inhibitor blocking liver fructose metabolism. In addition, S-700 (0.3–3 mg/kg) increased glucagon-like peptide 1 secretion, reduced food intake, and decreased body weight in Western Diet-fed rats without causing abnormal changes during the study. Finally, S-700 (3 mg/kg) completely prevented fructose-induced hypoglycemia in Aldob knockout mice, a model of hereditary fructose intolerance. Taken together, these results suggest that S-700 is a novel drug candidate for treating fructose-induced diseases.

Placental glucose transporter 1 and 3 gene expression in Monochorionic twin pregnancies with selective fetal growth restriction

BackgroundIn monochorionic twin (MC) gestations with selective fetal growth restriction (FGR), the discordant fetal growth usually is due to unequal placental sharing. Glucose, which is essential for oxidative metabolism in the growing placenta and fetus, is transferred from maternal blood by facilitated carrier-mediated diffusion via glucose transporters (GLUTs). How the GLUTs expression varies in the two placenta territories manifests discordant perfusion in MC twin pregnancy with selective FGR is unknown. This study evaluates the human placental GLUT1 and GLUT3 gene expression in MC twin gestations with selective FGR.MethodsMC twin pregnancy with selective FGR was defined as the presence of inter-twin birth weight discordance of > 25% and the smaller twin with a birth weight less than the 10th percentile in third trimester. Fetal umbilical artery Doppler was checked within 1 week before delivery in the two fetuses. An abnormal umbilical artery Doppler was defined as persistently absent or reverse end-diastolic flow (UA-AREDF). GLUT1, GLUT3 and HIF-1α gene expression were assayed in each twin’s placental territories. The inter-twin placental gene expression ratio was calculated as the placenta GLUTs or HIF-1α expression level of the selective FGR twin divided by expression level of the appropriate-for-gestational-age (AGA) cotwin. Higher gene expression ratio means elevated gene expression in the selective FGR twin’s placenta territory compared to AGA twin’s placenta territory.Results15 MC twin gestations with selective FGR including nine with normal (group 1) and six with abnormal selective FGR twin UA Doppler (group 2) were included into this study. The GLUT3 and HIF-1α gene expression are significantly elevated in selective FGR twin’s placenta territory in group 2 twin pregnancies (mean gene expression ratio as 2.23 and 1.65, p values as 0.015 and 0.045, respectively), but not in in group 1 twin pregnancies.ConclusionThe upregulation of placental GLUT3 gene expression in selective FGR fetus with abnormal UA Doppler may be due to hypo-perfusion which is mediated by up -regulation of HIF-1α gene expression.

Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs.

Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family.

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic "SP motif" and a conserved "A motif" stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6Å in complex with D-glucose and 3.7Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P.falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

Functional characterization and tissue localization of the facilitative glucose transporter GLUT12.

Facilitative glucose transporters (GLUTs) play crucial roles in glucose utilization and homeostasis. GLUT12 was initially isolated as a novel GLUT4-like transporter involved in insulin-dependent glucose transport. However, tissue distribution and biochemical properties of GLUT12 are not well understood. In this study, we investigated the basic kinetic properties and tissue distribution of GLUT12. Human GLUT12 and GLUT1 were overexpressed and purified using Ni-NTA column chromatography. Reconstituted proteoliposomes showed time-dependent d-glucose transport activity, which was inhibited by phloretin and dehydroascorbate. Dose dependence of glucose transport revealed a KM and Vmax values of 6.4 mM and 1.2 μmol/mg/min, respectively, indicating that GLUT12 is a high-affinity type GLUT. Glucose transport by GLUT12 was inhibited by ATP and glucose-1-phosphate, glucose-6-phosphate and disaccharides (properties similar to those of GLUT1). Indirect immunohistochemistry revealed the distribution of mouse GLUT12 in the apical region of distal tubules and collecting ducts in the kidney and epithelial cells of the jejunum. In addition to these cells, GLUT12 was present in chromaffin cells in the adrenal medulla, the anterior pituitary lobe, as well as the thyroid and pyloric glands. These tissue distributions suggest a unique function of GLUT12, besides that of an insulin-dependent glucose transport.

First Reports of Effects of Insulin, Human-like Insulin Receptors and Adapter Proteins in Acanthamoeba castellanii

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1-R) play key roles in growth, regulation of nutrient metabolism and carbohydrate homeostasis. Insulin-like molecules in prokaryotes and other early life have been reported. However, an account of metabolic effects of insulin, transcriptomic evidence of expression of glucose transporting channels (GLUT) and homology modelling of IR and IGF1-R like proteins in unicellular life-forms have yet to be established. Acanthamoeba spp. has existed for about 2 billion years and is one of the earliest mitochondriate unicellular eukaryotic cells on Earth. Despite Acanthamoeba spp. being grown in a medium called peptone-yeast-glucose (PYG) for over 50 years, the mechanism and regulation of glucose uptake by IR or IGF1-R molecules in this microbe has not yet been reported. Several methods were utilized to validate the effects of insulin on trophozoites of A. castellanii, including: growth assays with insulin, estimation of glucose and potassium (K+) entry into the cell, and histology showing anabolic effects on proteins. Bioinformatic computational tools and homology modeling demonstrated the involvement of IR like proteins, GLUT, and adapter proteins in mediating the IR cascade. Growth assays showed proliferative effects in a dose range of 2.98–5.97 µmol/mL of insulin. After insulin exposure, A. castellanii trophozoites displayed enhanced Periodic acid-Sciff (PAS) staining. Amino acid sequence similarities and homology modelling revealed ACA1_163470 in Acanthamoeba spp. to be a homolog of human-IR. Acanthamoeba protein ACA1_336150 shares similarities with IGF1-R. Additionally, some proteins like ACA1_060920 have attributes of GLUT like channels on homology modelling and show similarity with human GLUT. Knowledge of IR and insulin effects in Acanthamoeba spp. contributes to its biology and advances current understanding behind the evolution of IR and IGF1-R signalling cascade.

1734-P: High Dietary Fat Intake Exacerbates Insulin Resistance in a Type 2 Diabetes Genetic Mouse Model

GLUT4, an insulin-sensitive glucose transporter, plays a major role of postprandial glucose disposal. We previously reported that human GLUT4 promoter-driven insulin receptor knockout (GIRKO) mice have reduced insulin signaling in central and peripheral tissues and develop hyperglycemia, albeit at a lower rate in an age-dependent manner on normal chow diet. Long-term high fat diet (HFD) feeding is well established for causing adiposity gain, increased inflammation, and insulin resistance. We hypothesized that HFD feeding will exacerbate the insulin resistance and result in rapid progression to overt diabetes in GIRKO mice. To test this, we examined the physiological response of euglycemic GIRKO mice to HFD in a time course study. We used indirect calorimetry analysis and found that GIRKO mice showed differences in energy partitioning shortly after HFD feeding started. The majority of GIRKO mice cohorts had 5-fold higher circulating insulin and 1.5-fold elevated blood glucose after 1 month of HFD feeding. In addition to the hyperglycemia and hyperinsulinemia, GIRKO mice also exhibited impaired glucose tolerance and islet hyperplasia, suggesting extreme insulin resistance exceeded the compensatory increase of insulin production. After 3 months on HFD, GIRKO mice had increased relative liver size, elevated hepatic triglycerides, and reduced hepatic glycogen, demonstrating impaired hepatic insulin signaling to stimulate glycogen synthesis and inhibit gluconeogenesis. We also confirmed the increase of key hepatic gluconeogenic gene expression, which likely contributed to hyperglycemia. The rapid progression of metabolic defects of GIRKO mice on HFD is not due to excessive weight or adiposity gain. In conclusion, our studies of HFD-fed GIRKO mouse will offer insights in the role of the dietary contribution to diabetes disease progression without the confounding factor of increased adiposity. Disclosure A. Reilly: None. S. Yan: None. A.J. Loncharich: None. H. Ren: None. Funding Indiana University Diabetes and Obesity Research Training Program (T32DK064466); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK120772, R00DK098294); Indiana Diabetes Center (P30DK097512); Indiana Clinical and Translational Sciences Institute (UL1TR002529)

Identification of the fructose transporter GLUT5 (SLC2A5) as a novel target of nuclear receptor LXR

Fructose has become a major constituent of our modern diet and is implicated as an underlying cause in the development of metabolic diseases. The fructose transporter GLUT5 (SLC2A5) is required for intestinal fructose absorption. GLUT5 expression is induced in the intestine and skeletal muscle of type 2 diabetes (T2D) patients and in certain cancers that are dependent on fructose metabolism, indicating that modulation of GLUT5 levels could have potential in the treatment of these diseases. Using an unbiased screen for transcriptional control of the human GLUT5 promoter we identified a strong and specific regulation by liver X receptor α (LXRα, NR1H3). Using promoter truncations and site-directed mutagenesis we identified a functional LXR response element (LXRE) in the human GLUT5 promoter, located at −385 bp relative to the transcriptional start site (TSS). Finally, mice treated with LXR agonist T0901317 showed an increase in Glut5 mRNA and protein levels in duodenum and adipose tissue, underscoring the in vivo relevance of its regulation by LXR. Together, our findings show that LXRα regulates GLUT5 in mice and humans. As a ligand-activated transcription factor, LXRα might provide novel pharmacologic strategies for the selective modulation of GLUT5 activity in the treatment of metabolic disease as well as cancer.

Human placental GLUT1 glucose transporter expression and the fetal insulin-like growth factor axis in pregnancies complicated by diabetes

We have previously described regulation of syncytial GLUT1 glucose transporters by IGF-I. Despite this, it is not clear what signal regulates transplacental glucose transport. In this report we asked whether changes in GLUT1 expression and glucose transport activity in diabetic pregnancies were associated with alterations in the fetal IGF axis. Cord blood samples and paired syncytial microvillous and basal membranes were isolated from normal term pregnancies and pregnancies characterized by gestational diabetes type A2 (GDM A2) and pre-existing insulin-dependent diabetes mellitus (IDDM). Circulating IGF-I, basal membrane GLUT1 expression and glucose transporter activity were correlated with birth weight, but only in control, not diabetic groups. Basal membrane GLUT1 and transporter activity were correlated with IGF-I concentrations in control, but not diabetic groups. IGF binding protein (IGFBP) binding capacity showed a ≥50% reduction in the diabetic groups compared to control; both showed a higher level of free IGF-I. The absence of a correlation between birth weight and factors such as fetal IGF-I or GLUT1 expression in the diabetic groups suggests that IGF-I-stimulated effects may have reached a limiting threshold, such that further increases in IGF-I (or GLUT1) are without effect. These data support that fetal IGF-I acts as a fetal nutritional signal, modulating placental GLUT1 expression and birth weight via altered levels of fetal circulating IGFBPs. Diabetes appears to exert its effects on fetal and placental factors prior to the third trimester and, despite good glycemic control immediately prior to, and in the third trimester, these effects persist to term.