Identifying the transporters of different flavonoids in plants

We recently identified a new component of flavonoid transport pathways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seedling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT’s substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H+-ATPases and possible redundancy among the close homologues within the large Arabidopsis MATE family

Metabolic and cardiac adaptation to chronic pharmacologic blockade of facilitative glucose transport in murine dilated cardiomyopathy and myocardial ischemia

Abstract GLUT transgenic and knockout mice have provided valuable insight into the role of facilitative glucose transporters (GLUTs) in cardiovascular and metabolic disease, but compensatory physiological changes can hinder interpretation of these models. To determine whether adaptations occur in response to GLUT inhibition in the failing adult heart, we chronically treated TG9 mice, a transgenic model of dilated cardiomyopathy and heart failure, with the GLUT inhibitor ritonavir. Glucose tolerance was significantly improved with chronic treatment and correlated with decreased adipose tissue retinol binding protein 4 (RBP4) and resistin. A modest improvement in lifespan was associated with decreased cardiomyocyte brain natriuretic peptide (BNP) expression, a marker of heart failure severity. GLUT1 and −12 protein expression was significantly increased in left ventricular (LV) myocardium in ritonavir-treated animals. Supporting a switch from fatty acid to glucose utilization in these tissues, fatty acid transporter CD36 and fatty acid transcriptional regulator peroxisome proliferator-activated receptor α (PPARα) mRNA were also decreased in LV and soleus muscle. Chronic ritonavir also increased cardiac output and dV/dt-d in C57Bl/6 mice following ischemia-reperfusion injury. Taken together, these data demonstrate compensatory metabolic adaptation in response to chronic GLUT blockade as a means to evade deleterious changes in the failing heart

Improved De Novo Peptide Sequencing using LC Retention Time Information

Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) is an important tool in proteomics for identifying the peptides in a sample. Liquid chromatography temporally separates the peptides and tandem mass spectrometry analyzes the peptides, that elute one after another, by measuring their mass-to-charge ratios and the mass-to-charge ratios of their prefix and suffix fragments. De novo peptide sequencing is the problem of reconstructing the amino acid sequences of the analyzed peptide from this measurement data. While previous approaches solely consider the mass spectrum of the fragments for reconstructing a sequence, we propose to also exploit the information obtained from liquid chromatography. We study the problem of computing a sequence that is not only in accordance with the experimental mass spectrum, but also with the retention time of the separation by liquid chromatography. We consider three models for predicting the retention time of a peptide and develop algorithms for de novo sequencing for each model. An evaluation on experimental data from synthesized peptides for two of these models shows an improved performance compared to not using the chromatographic information.ISSN:1868-896

Expression and regulation of transmembrane transporters in healthy intestine and gastrointestinal diseases

Transmembrane transporters mediate energy dependent or independent translocation of drugs, potentially toxic compounds, and of various endogenous substrates such as bile acids and bilirubin across membranes. In this thesis the focus is on two classes of transporters, the ATPbinding cassette (ABC) transporters, which mediate ATP dependent transport and the solute carriers (SLC) which use electrochemical gradients for their transport. The transporters are expressed on membranes of cells of excretory organs (e.g. kidney, liver) and protective barriers (e.g. intestine, blood brain barrier) and influence therefore the absorption, distribution and elimination of compounds. They may reduce the intracellular concentration of drugs. Transport activity of transmembrane transporters in the intestine depends on the expression level and distribution along the intestine. Transport activity of transporters might be influenced by other factors such as genetic variations, which may present with a dysfunctional phenotype (e.g. single nucleotide polymorphisms; SNPs), or certain disease states, which might adaptively regulate transporter expression on the transcriptional and posttranscriptional level. The first aim of this thesis was a systematic site-specific analysis of the expression of several ABC transporters and solute carriers along the intestinal tract. Following that, regulation of hepatobiliary transporters in the human intestine during obstructive cholestasis was evaluated. Studies were performed in close collaboration with the Department of Gastroenterology (University Hospital of Basel). As described in Chapter 2, transporter mRNA expression was analyzed by real time PCR (Taqman), a method that was previously developed in our laboratory to quantify the expression of transporters using standard curves. Protein expression was assessed by immunohistochemistry, bile acids plasma concentrations were measured by capillary gas chromatography (U. Beuers, Munich). The results presented in this thesis include systematic site specific analysis of quantitative expression of the human multidrug resistance transporters, such MDR1 (ABCB1), breast cancer resistance transporter (BCRP; ABCG2) and multidrug resistance associated transporters (MRP1-5; ABCC1-5) as well as the apical sodium dependent bile acid transporter ASBT (SLC10A2) along the the intestinal tract. These data are shown in Chapter 3. As the mRNA expression levels of the investigated transporters change along the intestinal tract, these findings might be of interest to develop target strategies for orally administered drugs. Also, this systematic site specific analysis of MDR transporters serves as a preparation for a prospective clinical study in patients with Inflammatory Bowel Disease (IBD), which will investigate MDR transporter gene expression in intestinal biopsies (intestinal epithelial cells, intestinal macrophages) in newly diagnosed IBD patients compared to treatment refractory IBD patients, patients in remission and disease free controls. This study design will help to evaluate, if MDR transporters vary due to interindividual differences, inflammatory processes and/or pharmacological treatment and might serve as an explanation for patients with IBD not responding to drug treatment. Intestinal macrophages play a central role in the orchestration of innate immune response reactions in the gut. As anti-inflammatory as well as immunosuppresive drugs such as glucocorticoids, methotrexate, cyclosporine, 6-mercaptopurine and sulfasalazine, which all of them are used in the treatment of IBD patients, are substrates of MDR transporters, a method for isolation of CD14+ peripheral blood cells (monocytes), their ex vivo cultivation and differentiation into macrophages was established. In Chapter 4, first results demonstrate the influence of the differentiation process of monocytes into monocyte derived macrophages (MDM) and the stimulation of MDM with bacterial products (LPS) on MDR transporter expression. At present, the effect of budesonide, methotrexate, 6-mercaptopurine and sulfasalazine on the transporter expression in ex vivo cultivated human MDM is evaluated. Adaptive regulation of hepatobiliary transport systems during obstructive cholestasis with a disrupted enterohepatic circulation has been demonstrated in the intestine only for rodents before. The results presented in Chapter 3 showed, that ASBT, which contributes substantially to the enterohepatic circulation of bile acids by their reabsorption from the intestine, is adaptivelly regulated in the human duodenum during obstructive cholestasis. Our findings are of clinical importance as we have shown for the first time that ASBT is expressed in the human duodenum. These results may indicate species specific differences to rodents, and that changes in the ASBT gene expression can be measured in the duodenum during obstructive cholestasis. Adaptive regulation of ASBT in the intestine has clinical implications for the bile acid homeostasis and also for the lipid metabolism. BCRP mediates energy dependent efflux of drugs and potentially toxic compounds, and of various endogenous substrates such as bile acids. Here, expression of human BCRP mRNA was shown to be highest in the duodenum with a continuos decrease along the intestinal tract down to the rectum. BCRP mRNA and protein expression in the duodenum was found to be decreased during obstructive cholestasis when compared to control subjects and BCRP expression increased after reconstitution of bile flow. In consequence, reduced intestinal BCRP expression during obstructive cholestasis might influence the accumulation of bile acids, foodderived carcinogens and the pharmacokinetics of various drugs that are transported by BCRP. In an isolated project with the background of observed therapy resistance to antibiotics in the treatment of patients with chronic prostatitis, MDR expression in prostatic tissue in regard to inflammation was evaluated in 50 patients that underwent transurethral resection of prostate. In this study group, neither inflammation nor localization of inflammation in prostate tissue (acinar versus interstitial) influenced MDR transporter expression

HIV Protease Inhibitors Act as Competitive Inhibitors of the Cytoplasmic Glucose Binding Site of GLUTs with Differing Affinities for GLUT1 and GLUT4

The clinical use of several first generation HIV protease inhibitors (PIs) is associated with the development of insulin resistance. Indinavir has been shown to act as a potent reversible noncompetitive inhibitor of zero-trans glucose influx via direct interaction with the insulin responsive facilitative glucose transporter GLUT4. Newer drugs within this class have differing effects on insulin sensitivity in treated patients. GLUTs are known to contain two distinct glucose-binding sites that are located on opposite sides of the lipid bilayer. To determine whether interference with the cytoplasmic glucose binding site is responsible for differential effects of PIs on glucose transport, intact intracellular membrane vesicles containing GLUT1 and GLUT4, which have an inverted transporter orientation relative to the plasma membrane, were isolated from 3T3-L1 adipocytes. The binding of biotinylated ATB-BMPA, a membrane impermeable bis-mannose containing photolabel, was determined in the presence of indinavir, ritonavir, atazanavir, tipranavir, and cytochalasin b. Zero-trans 2-deoxyglucose transport was measured in both 3T3-L1 fibroblasts and primary rat adipocytes acutely exposed to these compounds. PI inhibition of glucose transport correlated strongly with the PI inhibition of ATB-BMPA/transporter binding. At therapeutically relevant concentrations, ritonavir was not selective for GLUT4 over GLUT1. Indinavir was found to act as a competitive inhibitor of the cytoplasmic glucose binding site of GLUT4 with a KI of 8.2 µM. These data establish biotinylated ATB-BMPA as an effective probe to quantify accessibility of the endofacial glucose-binding site in GLUTs and reveal that the ability of PIs to block this site differs among drugs within this class. This provides mechanistic insight into the basis for the clinical variation in drug-related metabolic toxicity

A novel FRET-based screen in high-throughput format to identify inhibitors of malarial and human glucose transporters

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z′ factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC(50)) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening

GLUT4, GLUT1, and GLUT8 are the dominant GLUT transcripts expressed in the murine left ventricle

BACKGROUND: The heart derives energy from a wide variety of substrates including fatty acids, carbohydrates, ketones, and amino acids. The healthy heart generates up to 30% of its ATP from glucose. Under conditions of cardiac injury or stress, the heart relies even more heavily on glucose as a source of fuel. Glucose is transported into the heart by members of the family of facilitative glucose transporters (GLUTs). While research examining the transport of glucose into the heart has primarily focused on the roles of the classical glucose transporters GLUT1 and GLUT4, little is known about the functions of more newly identified GLUT isoforms in the myocardium. METHODS: In this study the presence and relative RNA message abundance of each of the known GLUT isoforms was determined in left ventricular tissue from two commonly used inbred laboratory mouse strains (C57BL/6J and FVB/NJ) by quantitative real time PCR. Relative message abundance was also determined in GLUT4 null mice and in murine models of dilated and hypertrophic cardiomyopathy. RESULTS: GLUT4, GLUT1, and GLUT8 were found to be the most abundant GLUT transcripts in the normal heart, while GLUT3, GLUT10, and GLUT12 are present at relatively lower levels. Assessment of relative GLUT expression in left ventricular myocardium from mice with dilated cardiomyopathy revealed increased expression of GLUT1 with reduced levels of GLUT4, GLUT8, and GLUT12. Compensatory increase in the expression of GLUT12 was observed in genetically altered mice lacking GLUT4. CONCLUSIONS: Glucose transporter expression varies significantly among murine models of cardiac dysfunction and involves several of the class III GLUT isoforms. Understanding how these more newly identified GLUT isoforms contribute to regulating myocardial glucose transport will enhance our comprehension of the normal physiology and pathophysiology of the heart

The glucose transporter PfHT1 is an antimalarial target of the HIV protease inhibitor lopinavir

Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of Plasmodium species to existing therapies has increased the need for new therapeutic approaches. The Plasmodium glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population

GS-8374, a novel HIV protease inhibitor, does not alter glucose homeostasis in cultured adipocytes or in a healthy-rodent model system

Adverse effects induced by HIV protease inhibitors (PIs) are a significant factor in limiting their clinical success. PIs directly contribute to peripheral insulin resistance and alterations in lipid metabolism. GS-8374 is a novel PI with potent antiretroviral activity and a favorable resistance profile. Here we report on the potential of GS-8374 to adversely affect glucose and lipid homeostasis. Acute effects of GS-8374 and control PIs on glucose uptake and lipid accumulation were assessed in vitro in mouse OP9 and primary human adipocytes, respectively. GS-8374 and atazanavir showed no effect on insulin-stimulated deoxyglucose uptake, whereas ritonavir and lopinavir caused significant reductions. Similarly, in vitro lipid accumulation was not significantly affected in adipocytes treated with either GS-8374 or atazanavir. In euglycemic-hyperinsulinemic clamp experiments performed in rats during acute infusion of therapeutic levels of PIs, sustained serum GS-8374 levels of 8 μM had no effect on peripheral glucose disposal (similar to the findings for atazanavir). Comparable serum levels of lopinavir and ritonavir produced acute 19% and 53% reductions in in vivo glucose disposal, respectively. In conclusion, similar to atazanavir, but unlike ritonavir and lopinavir, GS-8374 neither affects insulin-stimulated glucose uptake in adipocytes in culture nor acutely alters peripheral glucose disposal in a rodent model system. These results dissociate the antiretroviral activity of GS-8374 from adverse effects on insulin sensitivity observed with some of the first-generation PIs and provide further support for the use of these experimental systems in the preclinical evaluation of novel PIs