Fructose Uptake Research Articles

Restoration of mRNA Expression of Solute Carrier Proteins in Liver of Diet-Induced Obese Mice by Metformin.

Metformin (MET), the most common medicine for type 2 diabetes (T2DM), improves insulin sensitivity by targeting the liver, intestine and other organs. Its impact on expression of the solute carrier (Slc) transporter genes have not been reported in the mechanism of insulin sensitization. In this study, we examined Slc gene expression in the liver and colon of diet-induced obese (DIO) mice treated with MET by transcriptomic analysis. There were 939 differentially expressed genes (DEGs) in the liver of DIO mice vs lean mice, which included 34 Slc genes. MET altered 489 DEGs in the liver of DIO mice, in which 23 were Slc genes. Expression of 20 MET-responsive Slc DEGs was confirmed by qRT-PCR, in which 15 Slc genes were altered in DIO mice and their expressions were restored by MET, including Slc2a10, Slc2a13, Slc5a9, Slc6a14, Slc7a9, Slc9a2, Slc9a3, Slc13a2, Slc15a2, Slc26a3, Slc34a2, Slc37a1, Slc44a4, Slc51b and Slc52a3. While, there were only 97 DEGs in the colon of DIO mice with 5 Slc genes, whose expression was not restored by MET. The data suggest that more genes were altered in the liver over the colon by the high fat diet (HFD). There were 20 Slc genes with alteration confirmed in the liver of DIO mice and 15 of them were restored by MET, which was associated with improvement of insulin sensitivity and obesity. The restoration may improve the uptake of glucose, amino acids, mannose, fructose, 1,5-anhydro-D-glucitol and bumetanide in hepatocytes of the liver of DIO mice. The study provides new insight into the mechanism of metformin action in insulin sensitization and obesity.

Inhibitory Effect of Tangeretin and Cardamonin on Human Intestinal SGLT1 Activity In Vitro and Blood Glucose Levels in Mice In Vivo.

Rapid postprandial blood glucose elevation can cause lifestyle-related diseases, such as type II diabetes. The absorption of food-derived glucose is primarily mediated by sodium/glucose cotransporter 1 (SGLT1). Moderate SGLT1 inhibition can help attenuate postprandial blood glucose elevation and prevent lifestyle-related diseases. In this study, we established a CHO cell line stably expressing human SGLT1 and examined the effects of phytochemicals on SGLT1 activity. Among the 50 phytochemicals assessed, tangeretin and cardamonin inhibited SGLT1 activity. Tangeretin and cardamonin did not affect the uptake of L-leucine, L-glutamate, and glycyl-sarcosine. Tangeretin, but not cardamonin, inhibited fructose uptake, suggesting that the inhibitory effect of tangeretin was specific to the monosaccharide transporter, whereas that of cardamonin was specific to SGLT1. Kinetic analysis suggested that the suppression of SGLT1 activity by tangeretin was associated with a reduction in Vmax and an increase in Km, whereas suppression by cardamonin was associated with a reduction in Vmax and no change in Km. Oral glucose tolerance tests in mice showed that tangeretin and cardamonin significantly suppressed the rapid increase in blood glucose levels. In conclusion, tangeretin and cardamonin were shown to inhibit SGLT1 activity in vitro and lower blood glucose level in vivo.

Phenolics-Rich Extracts of Dietary Plants as Regulators of Fructose Uptake in Caco-2 Cells via GLUT5 Involvement.

The latest data link the chronic consumption of large amounts of fructose present in food with the generation of hypertension and disturbances in carbohydrate and lipid metabolism, which promote the development of obesity, non-alcoholic fatty liver disease, insulin resistance, and type 2 diabetes. This effect is possible after fructose is absorbed by the small intestine cells and, to a lesser extent, by hepatocytes. Fructose transport is dependent on proteins from the family of glucose transporters (GLUTs), among which GLUT5 selectively absorbs fructose from the intestine. In this study, we examined the effect of four phenolic-rich extracts obtained from A. graveolens, B. juncea, and M. chamomilla on fructose uptake by Caco-2 cells. Extracts from B. juncea and M. chamomilla most effectively reduced fluorescent fructose analogue (NBDF) accumulation in Caco-2, as well as downregulated GLUT5 protein levels. These preparations were able to decrease the mRNA level of genes encoding transcription factors regulating GLUT5 expression-thioredoxin-interacting protein (TXNIP) and carbohydrate-responsive element-binding protein (ChREBP). Active extracts contained large amounts of apigenin and flavonols. The molecular docking simulation suggested that some of identified phenolic constituents can play an important role in the inhibition of GLUT5-mediated fructose transport.

A fructose/H+ symporter controlled by a LacI-type regulator promotes survival of pandemic Vibrio cholerae in seawater

The bacterium Vibrio cholerae can colonize the human intestine and cause cholera, but spends much of its life cycle in seawater. The pathogen must adapt to substantial environmental changes when moving between seawater and the human intestine, including different availability of carbon sources such as fructose. Here, we use in vitro experiments as well as mouse intestinal colonization assays to study the mechanisms used by pandemic V. cholerae to adapt to these environmental changes. We show that a LacI-type regulator (FruI) and a fructose/H+ symporter (FruT) are important for fructose uptake at low fructose concentrations, as those found in seawater. FruT is downregulated by FruI, which is upregulated when O2 concentrations are low (as in the intestine) by ArcAB, a two-component system known to respond to changes in oxygen levels. As a result, the bacteria predominantly use FruT for fructose uptake under seawater conditions (low fructose, high O2), and use a known fructose phosphotransferase system (PTS, Fpr) for fructose uptake under conditions found in the intestine. PTS activity leads to reduced levels of intracellular cAMP, which in turn upregulate virulence genes. Our results indicate that the FruT/FruI system may be important for survival of pandemic V. cholerae in seawater.

Genomic analysis of Leuconostoc citreum TR116 with metabolic reconstruction and the effects of fructose on gene expression for mannitol production

The species Leuconostoc citreum is often isolated from grain and vegetable fermentations such as sourdough, sauerkraut and kimchi. Lc. citreum has seen an increase in its use as a starter culture for various fermentations and food applications. The strain Lc. citreum TR116 has been applied previously in this laboratory aimed at sugar depletion through metabolism resulting in the reduction of fructose to mannitol, a polyol considered as a sweet carbohydrate. Besides reducing sugar, TR116 showed flavour modulating characteristics and contributes to the extension of microbial shelf life. In order to obtain a better understanding of this strain and to fully use its set of abilities, the genome of Lc. citreum TR116 was sequenced using the Illumina MiSeq, assembly with SPAdes and annotated by the Prokaryotic Genome Annotation Pipeline. Metabolic reconstruction was employed to elucidate carbohydrate, organic acid and amino acid metabolism in the strain. Of particular interest was the gene expression analysis ascertained the influence of fructose on the genes mdh and manX involved in the uptake of fructose and its conversion to mannitol. This investigation, the first in Lc. citreum, illustrates the metabolic processes involved in fermentation used by this strain and demonstrates that in the presence of fructose, expression of the genes mdh and manX is increased. The resulting transparency of the skill set of TR116 contributes highly to future functionalisation of food systems and food ingredients.

Grape Polyphenols Attenuate Diet-Induced Obesity and Hepatic Steatosis in Mice in Association With Reduced Butyrate and Increased Markers of Intestinal Carbohydrate Oxidation.

A Western Diet (WD) low in fiber but high in fats and sugars contributes to obesity and non-alcoholic fatty liver disease (NAFLD). Supplementation with grape polyphenols (GPs) rich in B-type proanthocyanidins (PACs) can attenuate symptoms of cardiometabolic disease and alter the gut microbiota and its metabolites. We hypothesized that GP-mediated metabolic improvements would correlate with altered microbial metabolites such as short chain fatty acids (SCFAs). To more closely mimic a WD, C57BL/6J male mice were fed a low-fiber diet high in sucrose and butterfat along with 20% sucrose water to represent sugary beverages. This WD was supplemented with 1% GPs (WD-GP) to investigate the impact of GPs on energy balance, SCFA profile, and intestinal metabolism. Compared to WD-fed mice, the WD-GP group had higher lean mass along with lower fat mass, body weight, and hepatic steatosis despite consuming more calories from sucrose water. Indirect and direct calorimetry revealed that reduced adiposity in GP-supplemented mice was likely due to their greater energy expenditure, which resulted in lower energy efficiency compared to WD-fed mice. GP-supplemented mice had higher abundance of Akkermansia muciniphila, a gut microbe reported to increase energy expenditure. Short chain fatty acid measurements in colon content revealed that GP-supplemented mice had lower concentrations of butyrate, a major energy substrate of the distal intestine, and reduced valerate, a putrefactive SCFA. GP-supplementation also resulted in a lower acetate:propionate ratio suggesting reduced hepatic lipogenesis. Considering the higher sucrose consumption and reduced butyrate levels in GP-supplemented mice, we hypothesized that enterocytes would metabolize glucose and fructose as a replacement energy source. Ileal mRNA levels of glucose transporter-2 (GLUT2, SLC2A2) were increased indicating higher glucose and fructose uptake. Expression of ketohexokinase (KHK) was increased in ileum tissue suggesting increased fructolysis. A GP-induced increase in intestinal carbohydrate oxidation was supported by: (1) increased gene expression of duodenal pyruvate dehydrogenase (PDH), (2) a decreased ratio of lactate dehydrogenase a (LDHa): LDHb in jejunum and colon tissues, and (3) decreased duodenal and colonic lactate concentrations. These data indicate that GPs protect against WD-induced obesity and hepatic steatosis by diminishing portal delivery of lipogenic butyrate and sugars due to their increased intestinal utilization.

Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum.

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

Targeting of GLUT5 for Transporter-Mediated Drug-Delivery Is Contingent upon Substrate Hydrophilicity.

Specific link between high fructose uptake and cancer development and progression highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5 in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents. Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and 48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.

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.

GLUT5 (SLC2A5) enables fructose-mediated proliferation independent of ketohexokinase

BackgroundFructose is an abundant source of carbon and energy for cells to use for metabolism, but only certain cell types use fructose to proliferate. Tumor cells that acquire the ability to metabolize fructose have a fitness advantage over their neighboring cells, but the proteins that mediate fructose metabolism in this context are unknown. Here, we investigated the determinants of fructose-mediated cell proliferation.MethodsLive cell imaging and crystal violet assays were used to characterize the ability of several cell lines (RKO, H508, HepG2, Huh7, HEK293T (293T), A172, U118-MG, U87, MCF-7, MDA-MB-468, PC3, DLD1 HCT116, and 22RV1) to proliferate in fructose (i.e., the fructolytic ability). Fructose metabolism gene expression was determined by RT-qPCR and western blot for each cell line. A positive selection approach was used to “train” non-fructolytic PC3 cells to utilize fructose for proliferation. RNA-seq was performed on parental and trained PC3 cells to find key transcripts associated with fructolytic ability. A CRISPR-cas9 plasmid containing KHK-specific sgRNA was transfected in 293T cells to generate KHK-/- cells. Lentiviral transduction was used to overexpress empty vector, KHK, or GLUT5 in cells. Metabolic profiling was done with seahorse metabolic flux analysis as well as LC/MS metabolomics. Cell Titer Glo was used to determine cell sensitivity to 2-deoxyglucose in media containing either fructose or glucose.ResultsWe found that neither the tissue of origin nor expression level of any single gene related to fructose catabolism determine the fructolytic ability. However, cells cultured chronically in fructose can develop fructolytic ability. SLC2A5, encoding the fructose transporter, GLUT5, was specifically upregulated in these cells. Overexpression of GLUT5 in non-fructolytic cells enabled growth in fructose-containing media across cells of different origins. GLUT5 permitted fructose to flux through glycolysis using hexokinase (HK) and not ketohexokinase (KHK).ConclusionsWe show that GLUT5 is a robust and generalizable driver of fructose-dependent cell proliferation. This indicates that fructose uptake is the limiting factor for fructose-mediated cell proliferation. We further demonstrate that cellular proliferation with fructose is independent of KHK.

Apolipoprotein A5 controls fructose-induced metabolic dysregulation in mice

Background and aimsWestern dietary habits are partially characterized by increased uptake of fructose, which contributes to metabolic dysregulation and associated liver diseases. For example, a diet enriched with fructose drives insulin resistance and non-alcoholic fatty liver disease (NAFLD). The molecular hubs that control fructose-induced metabolic dysregulation are poorly understood. Apolipoprotein A5 (apoA5) controls triglyceride metabolism with a putative role in hepatic lipid deposition. We explored apoA5 as a rheostat for fructose-induced hepatic and metabolic disease in mammals. Methods and resultsApoA5 knock out (−/−) and wildtype (wt) mice were fed with high fructose diet or standard diet for 10 weeks. Afterwards, we conducted a metabolic characterization by insulin tolerance test as well as oral glucose tolerance test. Additionally, hepatic lipid content as well as transcription patterns of key enzymes and transcription factors in glucose and lipid metabolism were evaluated. Despite comparable body weight, insulin sensitivity was significantly improved in high fructose diet fed apoA5 (−/−) when compared to wildtype mice on the same diet. In parallel, hepatic triglyceride content was significantly lower in apoA5 (−/−) mice than in wt mice. No difference was seen between apoA5 (−/−) and wt mice on a standard diet. ConclusionApoA5 is involved in fructose-induced metabolic dysregulation and associated hepatic steatosis suggesting that apoA5 may be a novel target to treat metabolic diseases.

Phosphorus deficiency changes carbon isotope fractionation and triggers exudate reacquisition in tomato plants

Plant roots are able to exude vast amounts of metabolites into the rhizosphere in response to phosphorus (P) deficiency. Causing noteworthy costs in terms of energy and carbon (C) for the plants. Therefore, it is suggested that exudates reacquisition by roots could represent an energy saving strategy of plants. This study aimed at investigating the effect of P deficiency on the ability of hydroponically grown tomato plants to re-acquire specific compounds generally present in root exudates by using 13C-labelled molecules. Results showed that P deficient tomato plants were able to take up citrate (+ 37%) and malate (+ 37%), particularly when compared to controls. While glycine (+ 42%) and fructose (+ 49%) uptake was enhanced in P shortage, glucose acquisition was not affected by the nutritional status. Unexpectedly, results also showed that P deficiency leads to a 13C enrichment in both tomato roots and shoots over time (shoots—+ 2.66‰, roots—+ 2.64‰, compared to control plants), probably due to stomata closure triggered by P deficiency. These findings highlight that tomato plants are able to take up a wide range of metabolites belonging to root exudates, thus maximizing C trade off. This trait is particularly evident when plants grew in P deficiency.

Fructose Metabolism and Regulation of Extracellular Matrix Protein Gene Expression in Activated Macrophages

Abstract Objectives Recently fructose has been linked to the development of Nonalcoholic Fatty Liver Disease (NAFLD), but the mechanisms behind the progression remain to be elucidated. Kupffer cells have been identified as regulators of hepatic inflammation and extracellular matrix (ECM) proteins. Activated macrophages are known to express tissue inhibitor of metalloproteinase (TIMP1), which inhibits matrix metalloproteinase 9 (MMP9) activity of reconstructing ECM which leads to fibrosis. In humans and mouse models, TIMP1 expression is positively correlated with the progression of NAFLD. Based on these findings, we hypothesize that fructose regulates TIMP1 gene expression in activated proinflammatory macrophages. Methods Using an in vitro model of J774 macrophages and primary Kupffer cells, cells were treated to induce an M1 phenotype in the presence of glucose or fructose. Cells were harvested for RNA and RT-PCR was conducted to measure ECM gene expression. Isolated Kupffer cells were collected from C57BL/6J mice fed a high fat diet (HFD) supplemented with 30% fructose or 30% glucose and analyzed for ECM expression. To examine fructose uptake and intra- and extracellular metabolites, mass spectrophotometry and nuclear magnetic resonance was conducted. Results Timp1 gene expression was significantly increased in J77.4 cells treated with fructose compared to glucose. Surprisingly, fructose treatment decreased Mmp9 gene expression. Likewise, fructose treatment increased TIMP1 protein expression in isolated Kupffer cells. In vivo, isolated hepatic macrophages from mice fed HF and high fructose diet had elevated Timp1 gene expression compared to mice fed high glucose diet. Extracellular levels of lactate decreased by 1.5-fold in fructose treated J77.4 cells compared to glucose. Metabolites involved in the TCA cycle and mitochondrial function were decreased when treated with fructose compared to glucose in non-activated macrophages. However, fructose treatment increased intracellular methanol and acetate levels in M1 macrophages compared to glucose. Conclusions Our data suggest that fructose upregulates Timp1 expression and possibly decreases mitochondrial function while increasing acetate and methanol production, identifying new mechanisms by which fructose drives the progression of NAFLD. Funding Sources Kenan Institute North Carolina University.

Metabolic engineering of carbohydrate metabolism systems in Corynebacterium glutamicum for improving the efficiency of l-lysine production from mixed sugar

The efficiency of industrial fermentation process mainly depends on carbon yield, final titer and productivity. To improve the efficiency of l-lysine production from mixed sugar, we engineered carbohydrate metabolism systems to enhance the effective use of sugar in this study. A functional metabolic pathway of sucrose and fructose was engineered through introduction of fructokinase from Clostridium acetobutylicum. l-lysine production was further increased through replacement of phosphoenolpyruvate-dependent glucose and fructose uptake system (PTSGlc and PTSFru) by inositol permeases (IolT1 and IolT2) and ATP-dependent glucokinase (ATP-GlK). However, the shortage of intracellular ATP has a significantly negative impact on sugar consumption rate, cell growth and l-lysine production. To overcome this defect, the recombinant strain was modified to co-express bifunctional ADP-dependent glucokinase (ADP-GlK/PFK) and NADH dehydrogenase (NDH-2) as well as to inactivate SigmaH factor (SigH), thus reducing the consumption of ATP and increasing ATP regeneration. Combination of these genetic modifications resulted in an engineered C. glutamicum strain K-8 capable of producing 221.3 ± 17.6 g/L l-lysine with productivity of 5.53 g/L/h and carbon yield of 0.71 g/g glucose in fed-batch fermentation. As far as we know, this is the best efficiency of l-lysine production from mixed sugar. This is also the first report for improving the efficiency of l-lysine production by systematic modification of carbohydrate metabolism systems.

Abstract P3-02-11: Topographical ues overlaid with fructose-like molecules to assess breast cancer recruitment in nanofiber scaffolds

Abstract Tumorigenesis is a complex process involving numerous cellular signaling cascades and environmental factors. While 2D cultures for stiffness measurements and more recently 3D cultures to demonstrate differences in cell structures have been reported, very little is known about the impact of sugars in cell recruitment. In this study, we report the fabrication of 3D-scaffolds with different morphologies obtained by electrospinning with fluorescent fructose-like molecular probes to study cancer cell proliferation and migration. Using a FDA approved, biocompatible and biodegradable polymer Polycaprolactone (PCL), we electrospun nanofiber scaffolds having random, aligned, and honeycomb morphologies. Scaffolds with similar morphology without the fructose mimicking analogs were fabricated and used as controls. The degradation rate of the scaffolds was also characterized to ensure long-term availability of probes and mechanical stability of PCL. Cell viability, cell morphology, formation of colonies with changes in morphology were investigated. The changes in biophysical properties of tumor microenvironment with change in morphology of the scaffolds were investigated. In vitro tests for proliferation, alignment and migration of normal breast epithelial cells (184B5), adenocarcinoma (MCF-7), pre-malignant (MCF10AneoT) and triple-negative (MDAMB231 on the scaffolds on days 1, 2, and 3 were carried out. The morphology of the scaffolds was characterized using FE-SEM; cell proliferation was assessed using CellTiter-Blue® Viability Assay; migration and cell-scaffold interactions were investigated using phalloidin for F-Actin and fructose response was determined by immunostaining for facilitative fructose transporter GLUT-5. Our data indicates that while topographical features affected cell adhesion and proliferation, cell lines that responded to the fructose-like probes tended to be more invasive. Furthermore, the preference to a specific scaffold was greatly altered by the presence of the probes with MDAMB231 showing least preference after 72 hours and pre-malignant AneoT showing highest preference. However, there was no significant difference in the cell numbers between scaffolds with probes and those without for the pre-malignant cells while this difference was noticeable in the control cell lines. Further investigation into specific response to glucose and fructose uptake through their major transporters GLUT2 and GLUT5 are currently ongoing. Citation Format: Carolynn Que, Samerender Nagam Hanumantharao, Brennan Vogl, Marina Tanasova, Smitha Rao. Topographical ues overlaid with fructose-like molecules to assess breast cancer recruitment in nanofiber scaffolds [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-11.

Hexose transport in Torulaspora delbrueckii: identification of Igt1, a new dual-affinity transporter.

Torulaspora delbrueckii is a yeast species receiving increasing attention from the biotechnology industry, with particular relevance in the wine, beer and baking sectors. However, little is known about its sugar transporters and sugar transport capacity, frequently a rate-limiting step of sugar metabolism and efficient fermentation. Actually, only one glucose transporter, Lgt1, has been characterized so far. Here we report the identification and characterization of a second glucose transporter gene, IGT1, located in a cluster, upstream of LGT1 and downstream of two other putative hexose transporters. Functional characterization of IGT1 in a Saccharomycescerevisiae hxt-null strain revealed that it encodes a transporter able to mediate uptake of glucose, fructose and mannose and established that its affinity, as measured by Km, could be modulated by glucose concentration in the medium. In fact, IGT1-transformed S. cerevisiae hxt-null cells, grown in 0.1% glucose displayed biphasic glucose uptake kinetics with an intermediate- (Km=6.5±2.0mM)and a high-affinity (Km=0.10±0.01mM)component, whereas cells grown in 2% glucose displayed monophasic kinetics with an intermediate-affinity (Km of 11.5±1.5mM).This work contributes to a better characterization of glucose transport in T. delbrueckii, with relevant implications for its exploitation in the food industry.

Glucose transporter expression and regulation following a fast in the ruby-throated hummingbird, Archilochus colubris.

Hummingbirds, subsisting almost exclusively on nectar sugar, face extreme challenges to blood sugar regulation. The capacity for transmembrane sugar transport is mediated by the activity of facilitative glucose transporters (GLUTs) and their localisation to the plasma membrane (PM). In this study, we determined the relative protein abundance of GLUT1, GLUT2, GLUT3 and GLUT5 via immunoblot using custom-designed antibodies in whole-tissue homogenates and PM fractions of flight muscle, heart and liver of ruby-throated hummingbirds (Archilochus colubris). The GLUTs examined were detected in nearly all tissues tested. Hepatic GLUT1 was minimally present in whole-tissue homogenates and absent win PM fractions. GLUT5 was expressed in flight muscles at levels comparable to those of the liver, consistent with the hypothesised uniquely high fructose uptake and oxidation capacity of hummingbird flight muscles. To assess GLUT regulation, we fed ruby-throated hummingbirds 1 mol l-1 sucrose ad libitum for 24 h followed by either 1 h of fasting or continued feeding until sampling. We measured relative GLUT abundance and concentration of circulating sugars. Blood fructose concentration in fasted hummingbirds declined (∼5 mmol l-1 to ∼0.18 mmol l-1), while fructose-transporting GLUT2 and GLUT5 abundance did not change in PM fractions. Blood glucose concentrations remained elevated in fed and fasted hummingbirds (∼30 mmol l-1), while glucose-transporting GLUT1 and GLUT3 in flight muscle and liver PM fractions, respectively, declined in fasted birds. Our results suggest that glucose uptake capacity is dynamically reduced in response to fasting, allowing for maintenance of elevated blood glucose levels, while fructose uptake capacity remains constitutively elevated promoting depletion of blood total fructose within the first hour of a fast.

Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is <10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.

Fructose co-ingestion to increase carbohydrate availability in athletes.

Carbohydrate availability is important to maximize endurance performance during prolonged bouts of moderate‐ to high‐intensity exercise as well as for acute post‐exercise recovery. The primary form of carbohydrates that are typically ingested during and after exercise are glucose (polymers). However, intestinal glucose absorption can be limited by the capacity of the intestinal glucose transport system (SGLT1). Intestinal fructose uptake is not regulated by the same transport system, as it largely depends on GLUT5 as opposed to SGLT1 transporters. Combining the intake of glucose plus fructose can further increase total exogenous carbohydrate availability and, as such, allow higher exogenous carbohydrate oxidation rates. Ingesting a mixture of both glucose and fructose can improve endurance exercise performance compared to equivalent amounts of glucose (polymers) only. Fructose co‐ingestion can also accelerate post‐exercise (liver) glycogen repletion rates, which may be relevant when rapid (<24 h) recovery is required. Furthermore, fructose co‐ingestion can lower gastrointestinal distress when relatively large amounts of carbohydrate (>1.2 g/kg/h) are ingested during post‐exercise recovery. In conclusion, combined ingestion of fructose with glucose may be preferred over the ingestion of glucose (polymers) only to help trained athletes maximize endurance performance during prolonged moderate‐ to high‐intensity exercise sessions and accelerate post‐exercise (liver) glycogen repletion.

Health outcomes of a high fructose intake: the importance of physical activity.

Fructose metabolism is generally held to occur essentially in cells of the small bowel, the liver, and the kidneys expressing fructolytic enzymes (fructokinase, aldolase B and a triokinase). In these cells, fructose uptake and fructolysis are unregulated processes, resulting in the generation of intracellular triose phosphates proportionate to fructose intake. Triose phosphates are then processed into lactate, glucose and fatty acids to serve as metabolic substrates in other cells of the body. With small oral loads, fructose is mainly metabolized in the small bowel, while with larger loads fructose reaches the portal circulation and is largely extracted by the liver. A small portion, however, escapes liver extraction and is metabolized either in the kidneys or in other tissues through yet unspecified pathways. In sedentary subjects, consumption of a fructose‐rich diet for several days stimulates hepatic de novo lipogenesis, increases intrahepatic fat and blood triglyceride concentrations, and impairs insulin effects on hepatic glucose production. All these effects can be prevented when high fructose intake is associated with increased levels of physical activity. There is also evidence that, during exercise, fructose carbons are efficiently transferred to skeletal muscle as glucose and lactate to be used for energy production. Glucose and lactate formed from fructose can also contribute to the re‐synthesis of muscle glycogen after exercise. We therefore propose that the deleterious health effects of fructose are tightly related to an imbalance between fructose energy intake on one hand, and whole‐body energy output related to a low physical activity on the other hand.