In vitro and in vivo comparison of MRI chemical exchange saturation transfer (CEST) properties between native glucose and 3-O-Methyl-D-glucose in a murine tumor model.

D-Glucose and 3-O-Methyl-D-glucose (3OMG) have been shown to provide contrast in magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) images. However, a systematic comparison between these two molecules has yet to be performed. The current study deals with the assessment of the effect of pH, saturation power level (B1 ) and magnetic field strength (B0 ) on the MRI-CEST contrast with the aim of comparing the in vivo CEST contrast detectability of these two agents in the glucoCEST procedure. Phosphate-buffered solutions of D-Glucose or 3OMG (20 mM) were prepared at different pH values and Z-spectra were acquired at several B1 levels at 37°C. In vivo glucoCEST images were obtained at 3 and 7 T over a period of 30 min after injection of D-Glucose or 3OMG (at doses of 1.5 or 3 g/kg) in a murine melanoma tumor model (n = 3-5 mice for each molecule, dose and B0 field). A markedly different pH dependence of CEST response was observed in vitro for D-Glucose and 3OMG. The glucoCEST contrast enhancement in the tumor region following intravenous administration (at the 3 g/kg dose) was comparable for both molecules: 1%-2% at 3 T and 2%-3% at 7 T. The percentage change in saturation transfer that resulted was almost constant for 3OMG over the 30-min period, whereas a significant increase was detected for D-Glucose. Our results show similar CEST contrast efficiency but different temporal kinetics for the metabolizable and the nonmetabolizable glucose derivatives in a tumor murine model when administered at the same doses.

Comparison of intestinal glucose flux and electrogenic current demonstrates two absorptive pathways in pig and one in Nile tilapia and rainbow trout.

The mucosal-to-serosal flux of 14C 3-O-methyl-d-glucose was compared against the electrogenic transport of d-glucose across ex vivo intestinal segments of Nile tilapia, rainbow trout, and pig in Ussing chambers. The difference in affinities (Km "fingerprints") between pig flux and electrogenic transport of glucose, and the absence of this difference in tilapia and trout, suggest two absorptive pathways in the pig and one in the fish species examined. More specifically, the total mucosal-to-serosal flux revealed a super high-affinity, high-capacity (sHa/Hc) total glucose transport system in tilapia; a super high-affinity, low-capacity (sHa/Lc) total glucose transport system in trout and a low-affinity, low-capacity (La/Lc) total glucose transport system in pig. Comparatively, electrogenic glucose absorption revealed similar Km in both fish species, with a super high-affinity, high capacity (sHa/Hc) system in tilapia; a super high-affinity/super low-capacity (sHa/sLc) system in trout; but a different Km fingerprint in the pig, with a high-affinity, low-capacity (Ha/Lc) system. This was supported by different responses to inhibitors of sodium-dependent glucose transporters (SGLTs) and glucose transporter type 2 (GLUT2) administered on the apical side between species. More specifically, tilapia flux was inhibited by SGLT inhibitors, but not the GLUT2 inhibitor, whereas trout lacked response to inhibitors. In contrast, the pig responded to inhibition by both SGLT and GLUT2 inhibitors with a higher expression of GLUT2. Altogether, it would appear that two pathways are working together in the pig, allowing it to have continued absorption at high glucose concentrations, whereas this is not present in both tilapia and trout.

Highly efficient and enantioselective syntheses of (2S,3R)-3-alkyl- and alkenylglutamates from Fmoc-protected Garner's aldehyde.

A 6-step enantioselective synthesis of (2S,3R)-3-alkyl/alkenylglutamates, including the biologically significant amino acid, (2S,3R)-3-methylglutamate, protected for Fmoc SPPS, is reported. Overall yields range from 52-65%. Key to the success of these syntheses was the development of a high-yielding 2-step synthesis of Fmoc Garner's aldehyde followed by a Horner-Wadsworth-Emmons reaction to give the corresponding Fmoc Garner's enoate in a 94% yield. The diastereoselective 1,4-addition of lithium dialkylcuprates to the Fmoc Garner's enoate was explored. Significant decomposition occurred when using lithium diethylcuprate and conditions previously reported for the 1,4-addition of lithium dialkylcuprates to Boc or Cbz-protected Garner's enoate. An optimization study of this reaction resulted in a robust set of conditions that addressed the shortcomings of previously reported conditions. Under these conditions, highly diastereoselective (> 20:1 in most cases) 1,4-addition reactions of lithium dialkyl/dialkenylcuprates to the Fmoc Garner's enoate were achieved in 76-99% yield. The resulting 1,4-addition products were easily converted into the Fmoc-(2S,3R)-3-alkyl/alkenylglutamates in two steps.

Low concentrations of 3-O-methylglucose improve post thaw recovery in cryopreserved bovine spermatozoa.

A number of studies have explored the use of membrane permeable (usually metabolizable) and membrane impermeable saccharides to protect cells in general, and sperm in particular during cryopreservation. Critical concentrations for protective levels of sugars frequently range between 50 mmol/L and 500 mmol/L, where efficacy is attributed to the sugar's membrane stabilizing and glass forming attributes and colligative effects that reduce intra- and extracellular salt concentrations during freezing. Many studies on bull sperm have demonstrated that both permeating and non-permeating sugars have negligible positive effects on post-thaw viability. Recently, however, a non-metabolizable sugar, 3-O-Methylglucose (3-OMG), was shown to protect hepatocytes during liver cryopreservation at 0.1-0.3 mol/L. Because glucose is readily transported into sperm, we hypothesized that 3-OMG could be a new class of cryoprotectant to explore in bull sperm. Here we present positive results demonstrating that 3-OMG improves post thaw viability in bull sperm, and that this effect is not likely due to improved glass forming capabilities. In particular, in experiment 1, 3-OMG was added to the Tris-egg yolk-glycerol base media at levels from 0 mmol/L to 200 mmol/L. Semen from four bulls was collected and diluted with one of the cryopreservation media, cooled, and frozen following industry standard practices. Motility and mitochondrial activity were negatively impacted when concentration of 3-OMG was more than 25 mmol/L. Therefore, we explored lower concentrations in experiment 2, where semen from eight bulls was used to evaluate concentrations 5 mmol/L, 15 mmol/L and 25 mmol/L of 3-OMG compared with control. Motility and progressive motility in 5 mmol/L 3-OMG and in the control were significantly higher than 15 mmol/L and 25 mmol/L 3-OMG, whereas mitochondrial activity and acrosome integrity in 5 mmol/L 3-OMG were significantly better than the control freezing medium. In experiment 3, to evaluate whether the improved effects of 3-OMG are due to its non-metabolizing property, or due to colligative effects, we compared post-thaw viability in semen from four bulls cryopreserved with 5 mmol/L glucose, sucrose, or 3-OMG. Motility and progressive motility was significantly improved in 3-OMG compared to glucose or sucrose groups which were comparable to the EY control. In conclusion, 3-OMG at a concentration of 5 mmol/L in Tris-egg yolk-glycerol medium improves the post thaw motility, progressive motility and viability of bull sperm. The mechanism of action is not understood but because the efficacy is maximal at low concentrations, it is not likely due to improved intra- or extracellular glass forming abilities and may demonstrate a different protective mechanism than was shown in hepatocytes.

CEST MRI of 3-O-methyl-D-glucose uptake and accumulation in brain tumors.

PURPOSE: 3-O-Methyl-D-glucose (3-OMG) is a nonmetabolizable structural analog of glucose that offers potential to be used as a CEST-contrast agent for tumor detection. Here, we explore it for CEST-detection of malignant brain tumors and compare it with D-glucose. METHODS: Glioma xenografts of a U87-MG cell line were implanted in five mice. Dynamic 3-OMG weighted images were collected using CEST-MRI at 11.7 T at a single offset of 1.2 ppm, showing the effect of accumulation of the contrast agent in the tumor, following an intravenous injection of 3-OMG (3 g/kg). RESULTS: Tumor regions showed higher enhancement as compared to contralateral brain. The CEST contrast enhancement in the tumor region ranged from 2.5-5.0%, while it was 1.5-3.5% in contralateral brain. Previous D-glucose studies of the same tumor model showed an enhancement of 1.5-3.0% and 0.5-1.5% in tumor and contralateral brain, respectively. The signal gradually stabilized to a value that persisted for the length of the scan. CONCLUSIONS: 3-OMG shows a CEST contrast enhancement that is approximately twice as much as that of D-glucose for a similar tumor line. In view of its suggested low toxicity and transport properties across the BBB, 3-OMG provides an option to be used as a nonmetallic contrast agent for evaluating brain tumors.

Maternal and fetal genomes interplay through phosphoinositol 3-kinase(PI3K)-p110α signaling to modify placental resource allocation.

Pregnancy success and life-long health depend on a cooperative interaction between the mother and the fetus in the allocation of resources. As the site of materno-fetal nutrient transfer, the placenta is central to this interplay; however, the relative importance of the maternal versus fetal genotypes in modifying the allocation of resources to the fetus is unknown. Using genetic inactivation of the growth and metabolism regulator, Pik3ca (encoding PIK3CA also known as p110α, α/+), we examined the interplay between the maternal genome and the fetal genome on placental phenotype in litters of mixed genotype generated through reciprocal crosses of WT and α/+ mice. We demonstrate that placental growth and structure were impaired and associated with reduced growth of α/+ fetuses. Despite its defective development, the α/+ placenta adapted functionally to increase the supply of maternal glucose and amino acid to the fetus. The specific nature of these changes, however, depended on whether the mother was α/+ or WT and related to alterations in endocrine and metabolic profile induced by maternal p110α deficiency. Our findings thus show that the maternal genotype and environment programs placental growth and function and identify the placenta as critical in integrating both intrinsic and extrinsic signals governing materno-fetal resource allocation.

Effects of starvation and short-term refeeding on gastric emptying and postprandial blood glucose regulation in adolescent girls with anorexia nervosa.

Postprandial glucose is reduced in malnourished patients with anorexia nervosa (AN), but the mechanisms and duration for this remain unclear. We examined blood glucose, gastric emptying, and glucoregulatory hormone changes in malnourished patients with AN and during 2 wk of acute refeeding compared with healthy controls (HCs). Twenty-two female adolescents with AN and 17 age-matched female HCs were assessed after a 4-h fast. Patients were commenced on a refeeding protocol of 2,400 kcal/day. Gastric emptying (13C-octanoate breath test), glucose absorption (3-O-methylglucose), blood glucose, plasma glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), insulin, C-peptide, and glucagon responses to a mixed-nutrient test meal were measured on admission and 1 and 2 wk after refeeding. HCs were assessed once. On admission, patients had slower gastric emptying, lower postprandial glucose and insulin, and higher glucagon and GLP-1 than HCs ( P < 0.05). In patients with AN, the rise in glucose (0-30 min) correlated with gastric emptying ( P < 0.05). With refeeding, postprandial glucose and 3-O-methylglucose were higher, gastric emptying faster, and baseline insulin and C-peptide less ( P < 0.05), compared with admission. After 2 wk of refeeding, postprandial glucose remained lower, and glucagon and GLP-1 higher, in patients with AN than HCs ( P < 0.05) without differences in gastric emptying, baseline glucagon, or postprandial insulin. Delayed gastric emptying may underlie reduced postprandial glucose in starved patients with AN; however, postprandial glucose and glucoregulatory hormone changes persist after 2 wk of refeeding despite improved gastric emptying. Future research should explore whether reduced postprandial glucose in AN is related to medical risk by examining associated symptoms alongside continuous glucose monitoring during refeeding.

3-O-Methyl-D-glucose mutarotation and proton exchange rates assessed by 13C, 1H NMR and by chemical exchange saturation transfer and spin lock measurements.

3-O-Methyl-D-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation rates of 3OMG were studied by 1H and 13C NMR. This information is essential in designing the in vivo CEST experiments. At room temperature, the ratio of α and ß 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium was 6 h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock at pH 6.14 and a temperature of 4 °C, were in the range of 360-670 s-1.

CEST MRI of 3-O-methyl-D-glucose on different breast cancer models.

PURPOSE: To test the ability of chemical exchange saturation transfer (CEST) MRI of 3-O-methyl-D-glucose (3OMG) to detect tumors in several breast cancer models of murine and human origin, for different routes of administration of the agent and to compare the method with glucoCEST and with 18 FDG-PET on the same animals. METHODS: In vivo CEST MRI experiments were performed with a 7T Biospec animal MRI scanner on implanted orthotopic mammary tumors of mice before and after administration of 3OMG. RESULTS: A marked 3OMG-CEST MRI contrast that was correlated with the administrated dose was obtained in different breast cancer models and by intravenous, intraperitoneal, and per os methods of administration. The most aggressive breast cancer model yielded the highest CEST contrast. 3OMG-CEST contrast reached its maximum at 20 min after administration and lasted for more than an hour, while that of glucose was lower and diminished after 20 min. 3OMG-CEST showed comparable results to that of FDG PET. CONCLUSION: The sensitivity of the 3OMG-CEST MRI method indicates its potential for the detection of tumors in the clinic. Magn Reson Med 79:1061-1069, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Small intestinal epithelial permeability to water-soluble nutrients higher in passerine birds than in rodents.

In the small intestine transcellular and paracellular pathways are implicated in water-soluble nutrient absorption. In small birds the paracellular pathway is quantitatively important while transcellular pathway is much more important in terrestrial mammals. However, there is not a clear understanding of the mechanistic underpinnings of the differences among taxa. This study was aimed to test the hypothesis that paracellular permeability in perfused intestinal segments is higher in passerine birds than rodents. We performed in situ intestinal perfusions on individuals of three species of passerine birds (Passer domesticus, Taeniopygia guttata and Furnarius rufus) and two species of rodents (Mus musculus and Meriones ungiculatus). Using radio-labelled molecules, we measured the uptake of two nutrients absorbed by paracellular and transcellular pathways (L-proline and 3-O-methyl-D-glucose) and one carbohydrate that has no mediated transport (L-arabinose). Birds exhibited ~2 to ~3 times higher L-arabinose clearance per cm2 epithelium than rodents. Moreover, paracellular absorption accounted for proportionally more of 3-O-methyl-D-glucose and L-proline absorption in birds than in rodents. These differences could be explained by differences in intestinal permeability and not by other factors such as increased retention time or higher intestinal nominal surface area. Furthermore, analysis of our results and all other existing data on birds, bats and rodents shows that insectivorous species (one bird, two bats and a rodent) had only 30% of the clearance of L-arabinose of non-insectivorous species. This result may be explained by weaker natural selection for high paracellular permeability in animal- than in plant-consumers. Animal-consumers absorb less sugar and more amino acids, whose smaller molecular size allow them to traverse the paracellular pathway more extensively and faster than glucose.

WZB117 (2-Fluoro-6-(m-hydroxybenzoyloxy) Phenyl m-Hydroxybenzoate) Inhibits GLUT1-mediated Sugar Transport by Binding Reversibly at the Exofacial Sugar Binding Site.

WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 µm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 µm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 ≫ GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.

Characterization of the transport activity of SGLT2/MAP17, the renal low-affinity Na+-glucose cotransporter.

The cotransporter SGLT2 is responsible for 90% of renal glucose reabsorption, and we recently showed that MAP17 appears to work as a required ß-subunit. We report in the present study a detailed functional characterization of human SGLT2 in coexpression with human MAP17 in Xenopus laevis oocytes. Addition of external glucose generates a large inward current in the presence of Na, confirming an electrogenic transport mechanism. At a membrane potential of -50 mV, SGLT2 affinity constants for glucose and Na are 3.4 ± 0.4 and 18 ± 6 mM, respectively. The change in the reversal potential of the cotransport current as a function of external glucose concentration clearly confirms a 1:1 Na-to-glucose transport stoichiometry. SGLT2 is selective for glucose and α-methylglucose but also transports, to a lesser extent, galactose and 3-O-methylglucose. SGLT2 can be inhibited in a competitive manner by phlorizin (Ki = 31 ± 4 nM) and by dapagliflozin (Ki = 0.75 ± 0.3 nM). Similarly to SGLT1, SGLT2 can be activated by Na, Li, and protons. Pre-steady-state currents for SGLT2 do exist but are small in amplitude and relatively fast (a time constant of ~2 ms). The leak current defined as the phlorizin-sensitive current in the absence of substrate was extremely small in the case of SGLT2. In summary, in comparison with SGLT1, SGLT2 has a lower affinity for glucose, a transport stoichiometry of 1:1, very small pre-steady-state and leak currents, a 10-fold higher affinity for phlorizin, and an affinity for dapagliflozin in the subnanomolar range.

Regulation of GLUT4 activity in myotubes by 3-O-methyl-d-glucose.

The rate of glucose influx to skeletal muscles is determined primarily by the number of functional units of glucose transporter-4 (GLUT4) in the myotube plasma membrane. The abundance of GLUT4 in the plasma membrane is tightly regulated by insulin or contractile activity, which employ distinct pathways to translocate GLUT4-rich vesicles from intracellular compartments. Various studies have indicated that GLUT4 intrinsic activity is also regulated by conformational changes and/or interactions with membrane components and intracellular proteins in the vicinity of the plasma membrane. Here we show that the non-metabolizable glucose analog 3-O-methyl-d-glucose (MeGlc) augmented the rate of hexose transport into myotubes by increasing GLUT4 intrinsic activity without altering the content of the transporter in the plasma membrane. This effect was not a consequence of ATP depletion or hyperosmolar stress and did not involve Akt/PKB or AMPK signal transduction pathways. MeGlc reduced the inhibitory potency (increased Ki) of indinavir, a selective inhibitor of GLUT4, in a dose-dependent manner. Kinetic analyses indicate that MeGlc induced changes in GLUT4 or GLUT4 complexes within the plasma membrane, which enhanced the hexose transport activity and reduced the potency of indinavir inhibition. Finally, we present a simple kinetic analysis for screening and discovering low molecular weight compounds that augment GLUT4 activity.

Use of D-glucose-fenpiclonil conjugate as a potent and specific inhibitor of sucrose carriers.

Until now, specific inhibitors of sucrose carriers were not available. This led us to study the properties of the recently synthesized D-glucose-fenpiclonil conjugate (D-GFC). This large amphiphilic glucoside exhibited an extremely low phloem systemicity in contrast to L-amino acid-fenpiclonil conjugates. Using Ricinus seedlings, the effect of D-GFC on 0.5 mM [14C]sucrose (Suc), 3-O-[3H]methylglucose, and [3H]glutamine uptake by cotyledon tissues was compared with that of p-chloromercuribenzenesulfonic acid (PCMBS). D-GFC dramatically inhibited H+-Suc symport at the same concentrations as PCMBS (0.5 and 1 mM), but in contrast to the thiol reagent, it did not affect 3-O-methylglucose and glutamine transport, nor the acidification of the incubation medium by cotyledon tissues. Similarly, 0.5 mM D-GFC inhibited active Suc uptake by Vicia faba leaf tissues and by Saccharomyces cerevisiae cells transformed with AtSUC2, a gene involved in Suc phloem loading in Arabidopsis, by approximately 80%. The data indicated that D-GFC was a potent inhibitor of Suc uptake from the endosperm and of Suc phloem loading. It is the first chemical known to exhibit such specificity, at least in Ricinus, and this property permitted the quantification of the two routes involved in phloem loading of endogenous sugars after endosperm removal.

Metformin reduces the rate of small intestinal glucose absorption in type 2 diabetes.

In rodents, metformin slows intestinal glucose absorption, potentially increasing exposure of the distal gut to glucose to enhance postprandial glucagon-like peptide-1 (GLP-1) secretion. We evaluated the effects of metformin on serum 3-O-methylglucose (3-OMG; a marker of glucose absorption) and plasma total GLP-1 concentrations during a standardized intraduodenal infusion of glucose and 3-OMG in patients with type 2 diabetes. A total of 12 patients, treated with metformin 850 mg twice daily or placebo for 7 days each in a double-blind, randomized, crossover design (14 days' washout between treatments), were evaluated on days 5 or 8 of each treatment (6 subjects each). On each study day, 30 minutes after ingesting 850 mg metformin or placebo, patients received an infusion of glucose (60 g + 5 g 3-OMG, dissolved in water to 240 mL) via an intraduodenal catheter over the course of 120 minutes. Compared with placebo, metformin was associated with lower serum 3-OMG ( P < .001) and higher plasma total GLP-1 ( P = .003) concentrations. The increment in plasma GLP-1 after metformin vs placebo was related to the reduction in serum 3-OMG concentrations ( P = .019). Accordingly, metformin inhibits small intestinal glucose absorption, which may contribute to augmented GLP-1 secretion in type 2 diabetes.

Intestinal paracellular absorption is necessary to support the sugar oxidation cascade in nectarivorous bats.

We made the first measurements of the capacity for paracellular nutrient absorption in intact nectarivorous bats. Leptonycteris yerbabuenae (20 g mass) were injected with or fed inert carbohydrate probes L-rhamnose and D(+)-cellobiose, which are absorbed exclusively by the paracellular route, and 3-O-methyl-D-glucose (3OMD-glucose), which is absorbed both paracellularly and transcellularly. Using a standard pharmacokinetic technique, we collected blood samples for 2 h after probe administration. As predicted, fractional absorption (f) of paracellular probes declined with increasing Mr in the order of rhamnose (f=0.71)>cellobiose (f=0.23). Absorption of 3OMD-glucose was complete (f=0.85; not different from unity). Integrating our data with those for glucose absorption and oxidation in another nectarivorous bat, we conclude that passive paracellular absorption of glucose is extensive in nectarivorous bat species, as in other bats and small birds, and necessary to support high glucose fluxes hypothesized for the sugar oxidation cascade.

Cryopreservation of human spermatozoa with minimal non-permeable cryoprotectant.

Cryopreservation of human spermatozoa is a commonly used technique in assisted reproduction, however freezing low concentrations of sperm while maintaining adequate post-thaw motility remains a challenge. In an effort to optimize post-thaw motility yields, low volumes of human sperm were frozen in polyimide-coated fused silica micro-capillaries using 0.065 M, 0.125 M, 0.25 M, or 0.5 M trehalose as the only cryoprotectant. Micro-capillaries were either initially incubated in liquid nitrogen vapor before plunging into liquid nitrogen, or directly plunged into liquid nitrogen. Post thaw sperm counts and motility were estimated. Spermatozoa that were initially incubated in liquid nitrogen vapor had greater post thaw motility than those plunged immediately into liquid nitrogen independent of trehalose concentration. The protective effect of 0.125 M d-glucose, 3-O-methyl-d-glucopyranose, trehalose, sucrose, raffinose, or stachyose were evaluated individually. Trehalose and sucrose were the most effective cryoprotectants, recovering 69.0% and 68.9% of initial sperm motility, respectively.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

Selective sodium-dependent glucose transporter 1 inhibitors block glucose absorption and impair glucose-dependent insulinotropic peptide release.

GSK-1614235 and KGA-2727 are potent, selective inhibitors of the SGLT1 sodium-dependent glucose transporter. Nonclinical (KGA-2727) and clinical (GSK-1614235) trials assessed translation of SGLT1 inhibitor effects from rats to normal human physiology. In rats, KGA-2727 (0.1 mg/kg) or vehicle was given before oral administration of 3-O-methyl-α-d-glucopyranose (3-O-methylglucose, 3-OMG) containing 3-[3H]OMG tracer. Tracer absorption and distribution were assessed from plasma, urine, and fecal samples. SGLT1 inhibition reduced urine 3-OMG recovery and increased fecal excretion. SGLT1 inhibitor effects on plasma glucose, insulin, gastric inhibitory peptide (GIP), and glucagon-like peptide-1 (GLP-1) concentrations were also measured during a standard meal. Incremental glucose, insulin, and GIP concentrations were decreased, indicating downregulation of ß-cell and K cell secretion. Minimal effects were observed in the secretion of the L cell product, GLP-1. With the use of a three-way, crossover design, 12 healthy human subjects received placebo or 20 mg GSK-1614235 immediately before or after a meal. Five minutes into the meal, 3-OMG was ingested. Postmeal dosing had little impact, yet premeal dosing delayed and reduced 3-OMG absorption, with an AUC0-10 of 231±31 vs. 446±31 µg·h(-1)·ml(-1), for placebo. Recovery of tracer in urine was 1.2±0.7 g for premeal dosing and 2.2±0.1 g for placebo. Incremental concentrations of insulin, C-peptide, and GIP were reduced for 2 h with premeal GSK-1614235. Total GLP-1 concentrations were significantly increased, and a trend for increased peptide YY (PYY) was noted. SGLT1 inhibitors block intestinal glucose absorption and reduce GIP secretion in rats and humans, suggesting SGLT1 glucose transport is critical for GIP release. Conversely, GLP-1 and PYY secretion are enhanced by SGLT1 inhibition in humans.

Remodeling of glucose metabolism precedes pressure overload-induced left ventricular hypertrophy: review of a hypothesis.

When subjected to pressure overload, the ventricular myocardium shifts from fatty acids to glucose as its main source for energy provision and frequently increases its mass. Here, we review the evidence in support of the concept that metabolic remodeling, measured as an increased myocardial glucose uptake using dynamic positron emission tomography (PET) with the glucose analogue 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG), precedes the onset of left ventricular hypertrophy (LVH) and heart failure. Consistent with this, early intervention with propranolol, which attenuates glucose uptake, prevents the maladaptive metabolic response and preserves cardiac function in vivo. We also review ex vivo studies suggesting a link between dysregulated myocardial glucose metabolism, intracellular accumulation of glucose 6-phosphate (G6P) and contractile dysfunction of the heart. G6P levels correlate with activation of mTOR (mechanistic target of rapamycin) and endoplasmic reticulum stress. This sequence of events could be prevented by pretreatment with rapamycin (mTOR inhibition) or metformin (enzyme 5'-AMP-activated protein kinase activation). In conclusion, we propose that metabolic imaging with FDG PET may provide a novel approach to guide the treatment of patients with hypertension-induced LVH.