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We examined whether short-term treatment with a thiazolidinedione improves insulin sensitivity in non-obese but insulin-resistant subjects and whether this is associated with an improvement in dysregulated adipose tissue (reduced expression of IRS-1, GLUT4, PPARgamma co-activator 1 and markers of terminal differentiation) that we have previously documented to be associated with insulin resistance. Ten non-diabetic subjects, identified as having low IRS-1 and GLUT-4 protein in adipose cells as markers of insulin resistance, underwent 3 weeks of treatment with pioglitazone. The euglycaemic-hyperinsulinaemic clamp technique was used to measure insulin sensitivity before and after treatment. Serum samples were analysed for glucose, insulin, lipids, total and high-molecular-weight (HMW) adiponectin levels. Biopsies from abdominal subcutaneous adipose tissue were taken, cell size measured, mRNA and protein extracted and quantified using real-time RT-PCR and Western blot. Insulin sensitivity was improved after 3 weeks treatment and circulating total as well as HMW adiponectin increased in all subjects, while no effect was seen on serum lipids. In the adipose cells, gene and protein expression of IRS-1 and PPARgamma co-activator 1 remained unchanged, while adiponectin, adipocyte P 2, uncoupling protein 2, GLUT4 and liver X receptor-alpha increased. Insulin-stimulated tyrosine phosphorylation and p-ser-PKB/Akt increased, while no significant effect of thiazolidinedione treatment was seen on the inflammatory status of the adipose tissue in these non-obese subjects. Short-term treatment with pioglitazone improved insulin sensitivity in the absence of any changes in circulating NEFA or lipid levels. Several markers of adipose cell differentiation, previously shown to be reduced in insulin resistance, were augmented, supporting the concept that insulin resistance in these individuals is associated with impaired terminal differentiation of the adipose cells.

Impact of Aerobic Exercise Training on Age-Related Changes in Insulin Sensitivity and Muscle Oxidative Capacity Kevin R. Short 1 , Janet L. Vittone 2 , Maureen L. Bigelow 1 , David N. Proctor 3 , Robert A. Rizza 1 , Jill M. Coenen-Schimke 1 and K. Sreekumaran Nair 1 1 Department of Internal Medicine, Division of Endocrinology, Mayo Clinic, Rochester, Minnesota 2 Department of General Internal Medicine, Mayo Clinic, Rochester, Minnesota 3 Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota Address correspondence and reprint requests to K. Sreekumaran Nair, PhD, Endocrinology Research Unit, 5–194 Joseph, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. E-mail: nair.sree{at}mayo.edu Abstract Insulin resistance increases and muscle oxidative capacity decreases during aging, but lifestyle changes—especially physical activity—may reverse these trends. Here we report the effect of a 16-week aerobic exercise program ( n = 65) or control activity ( n = 37) performed by men and women aged 21–87 years on insulin sensitivity and muscle mitochondria. Insulin sensitivity, measured by intravenous glucose tolerance test, decreased with age ( r = −0.32) and was related to abdominal fat content ( r = −0.65). Exercise increased peak oxygen uptake ( V o 2peak ; 10%), activity of muscle mitochondrial enzymes (citrate synthase and cytochrome c oxidase, 45–76%) and mRNA levels of mitochondrial genes (COX4, ND4, both 66%) and genes involved in mitochondrial biogenesis (PGC-1α, 55%; NRF-1, 15%; TFAM, 85%). Exercise also increased muscle GLUT4 mRNA and protein (30–52%) and reduced abdominal fat (5%) and plasma triglycerides (25%). None of these changes were affected by age. In contrast, insulin sensitivity improved in younger people but not in middle-aged or older groups. Thus, the muscle mitochondrial response to 4 months of aerobic exercise training was similar in all age-groups, although the older people did not have an improvement in insulin sensitivity. GCRC, General Clinical Research Center NRF, nuclear respiratory factor PGC-1α, PPAR-γ coactivator 1α PPAR, peroxisome proliferator–activated receptor TFAM, mitochondrial transcription factor A Vo2peak, peak oxygen uptake SI, insulin sensitivity Footnotes Accepted May 1, 2003. Received October 31, 2002. Revision received May 1, 2003. DIABETES

The X-ray crystal structure is presented of a seven-transmembrane eukaryotic SWEET glucose transporter, revealing the link between seven-transmembrane eukaryotic SWEETs and their three-transmembrane bacterial homologues and providing insight into eukaryotic sugar transport mechanisms. Structure of a SWEET sugar transporter SWEET sugar transporters are involved in various processes in plants and in glucose transport in animals. The authors report the first X-ray crystal structure of a eukaryotic SWEET glucose transporter, a vacuolar glucose transporter from rice. The structure (of the inward-open state) shows that this transporter forms homomeric trimers. It contains seven transmembrane helices — in contrast to the three helices reported for bacterial homologues — suggesting a molecular basis for understanding functional cross-talk and coupling of SWEET transporters. Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs 1 , 2 , 3 , 4 , 5 . SWEETs carry mono- and disaccharides 6 across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion 7 , phloem loading for long distance translocation 8 , pollen nutrition 9 , and seed filling 10 . Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells 3 , 11 , 12 . The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection 13 . Here we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice ( Oryza sativa ), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Insights into the structure–function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter–drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR–Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC–drug associations for a number of clinically relevant compounds. A set of CRISPR–Cas9-based genetic screens in a haploid human cell line identifies more than 200 gene–drug associations involving solute carriers (SLCs), transporters important for the uptake and activity of cytotoxic drugs.

Sugar transport across tonoplasts is essential for maintaining cellular sugar homeostasis and metabolic balance in plant cells. It remains unclear, however, how this process is regulated among different classes of sugar transporters. Here, we identified a tonoplast H⁺/glucose symporter, MdERDL6-1, from apples, which was highly expressed in fruits and exhibited expression patterns similar to those of the tonoplast H⁺/sugar antiporters MdTST1 and MdTST2. Overexpression of MdERDL6-1 unexpectedly increased not only glucose (Glc) concentration but also that of fructose (Fru) and sucrose (Suc) in transgenic apple and tomato leaves and fruits. RNA sequencing (RNA-seq) and expression analyses showed an upregulation of TST1 and TST2 in the transgenic apple and tomato lines overexpressing MdERDL6-1. Further studies established that the increased sugar concentration in the transgenic lines correlated with up-regulation of TST1 and TST2 expression. Suppression or knockout of SlTST1 and SlTST2 in the MdERDL6-1–overexpressed tomato background reduced or abolished the positive effect of MdERDL6-1 on sugar accumulation, respectively. The findings demonstrate a regulation of TST1 and TST2 by MdERDL6-1, inwhich Glc exported by MdERDL6-1 from vacuole up-regulates TST1 and TST2 to import sugars from cytosol to vacuole for accumulation to high concentrations. The results provide insight into the regulatory mechanism of sugar accumulation in vacuoles mediated by the coordinated action of two classes of tonoplast sugar transporters.

Microbial pathogens strategically acquire metabolites from their hosts during infection. Here we show that the host can intervene to prevent such metabolite loss to pathogens. Phosphorylation-dependent regulation of sugar transport protein 13 (STP13) is required for antibacterial defense in the plant Arabidopsis thaliana. STP13 physically associates with the flagellin receptor flagellin-sensitive 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1). BAK1 phosphorylates STP13 at threonine 485, which enhances its monosaccharide uptake activity to compete with bacteria for extracellular sugars. Limiting the availability of extracellular sugar deprives bacteria of an energy source and restricts virulence factor delivery. Our results reveal that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection. Competition for sugar thus shapes host-pathogen interactions.

All known d -xylose transporters are competitively inhibited by d -glucose, which is one of the major reasons hampering simultaneous fermentation of d -glucose and d -xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant d -xylose transporters that are insensitive to the presence of d -glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the d -xylose transporter XylE from Escherichia coli , both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent d -glucose but not d -xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of d -glucose negatively affected transport of both d -glucose and d -xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for d -xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose–xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.

Two high-affinity proton-dependent transporters of glucosinolates have been identified in Arabidopsis and termed GTR1 and GTR2; these transporters are essential for transporting glucosinolates to seeds, offering a means to control the allocation of defence compounds in a tissue-specific manner, which may have agricultural biotechnology implications. Engineering more nutritional crops Glucosinolates are important plant defence compounds. They are synthesized in various tissues and then translocated to the seeds, where they accumulate. In this study, Barbara Halkier and colleagues examine the molecular basis of this long-distance transport process. They identify two high-affinity, proton-dependent glucosinolate-specific transporters in Arabidopsis , termed GTR1 and GTR2. These transporters control the loading of glucosinolates from the apoplast into the phloem. The authors' specific and complete elimination of glucosinolates from Arabidopsis seeds, combined with the compounds' retention in vegetative tissues, establishes transport engineering as a potential approach for eliminating anti-nutritional natural products in high-value crops. In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value 1 , as demonstrated in the plant model Arabidopsis , in which the major defence compounds, glucosinolates 2 , are translocated to seeds on maturation 3 . The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.

The glucose transporter GLUT1 catalyses facilitative diffusion of glucose into erythrocytes and is responsible for glucose supply to the brain and other organs. Dysfunctional mutations may lead to GLUT1 deficiency syndrome, whereas overexpression of GLUT1 is a prognostic indicator for cancer. Despite decades of investigation, the structure of GLUT1 remains unknown. Here we report the crystal structure of human GLUT1 at 3.2 Å resolution. The full-length protein, which has a canonical major facilitator superfamily fold, is captured in an inward-open conformation. This structure allows accurate mapping and potential mechanistic interpretation of disease-associated mutations in GLUT1. Structure-based analysis of these mutations provides an insight into the alternating access mechanism of GLUT1 and other members of the sugar porter subfamily. Structural comparison of the uniporter GLUT1 with its bacterial homologue XylE, a proton-coupled xylose symporter, allows examination of the transport mechanisms of both passive facilitators and active transporters. The structure of human GLUT1 in an inward-open conformation is reported; access to the structure of the human protein, instead of just a bacterial homologue, made it possible to map (inactivating) mutations associated with GLUT1 deficiency syndrome onto the structure. Human GLUT1 glucose transporter structure GLUT1 is a membrane protein that is responsible for the uptake of glucose into erythrocytes and other cells. The structure of a proton-coupled xylose symporter that is a bacterial homologue of GLUT1 has been reported previously and here Nieng Yan and colleagues report the structure of human GLUT1 in an inward-open conformation. Having access to the structure of the human protein, the authors were able to map inactivating mutations associated with GLUT1 deficiency syndrome — also known as De Vivo syndrome — onto their structure. Because elevated expression levels of GLUT1 have been observed in several cancer types, access to this structure may facilitate the development of new anticancer agents.

Sucrose (Suc) is one of the most important types of sugars in plants, serving inter alia as a long-distance transport molecule, a carbon and energy storage compound, an osmotically active solute, and fuel for many anabolic reactions. Suc biosynthesis and degradation pathways are well known; however, the regulation of Suc intracellular distribution is poorly understood. In particular, the cellular function of chloroplast Suc reserves and the transporters involved in accumulating these substantial Suc levels remain uncharacterized. Here, we characterize the plastidic sugar transporter (pSuT) in Arabidopsis (Arabidopsis thaliana), which belongs to a subfamily of the monosaccharide transporter-like family. Transport analyses with yeast cells expressing a truncated, vacuole-targeted version of pSuT indicate that both glucose and Suc act as substrates, and nonaqueous fractionation supports a role for pSuT in Suc export from the chloroplast. The latter process is required for a correct transition from vegetative to reproductive growth and influences inflorescence architecture. Moreover, pSuT activity affects freezing-induced electrolyte release. These data further underline the central function of the chloroplast for plant development and the modulation of stress tolerance.