Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues

An indicator dilution technique with ²²Na⁺ as the intravascular marker was used to measure unidirectional transport of d-[6-³H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, α-d-glucose has higher affinity for the carrier than does β-d-glucose.A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

The action of inhibitors of sugar transport phlorizin, phloretin and cytochalasin B in model systems

Phlorizin, phloretin and cytochalasin B are known to be specific sugar transport inhibitors. A study was made of their effects on the carbohydrate-protein interaction in solution as a model system for examining the initial steps of sugar membrane transport. Glycogen precipitation by concanavalin A is inhibited only by alpha-methylmannoside, whereas both phlorizin and phloretin inhibit interactions between hexokinase and glucose, and between glucose-6-phosphate dehydrogenase and glucose-6-phosphate. Cytochalasin B was found to exert no effect on both the concanavalin A--glycogen interaction and the enzyme reactions investigated. The data obtained in the model system examination may suggest that the sites of glucose and cytochalasin binding are, respectively, spatially uncoupled.     

Inhibition of brain mitochondrial Ca2+ transport by amiloride analogues

Rat brain mitochondrial Ca2+ uptake and release were examined in the presence of amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)-pyrazinecarboxamide) and nineteen amiloride analogues. Amiloride, an inhibitor of Na+-Ca2+ exchange in plasmalemma membranes, did not affect energy-dependent Ca2+ uptake, whereas several other analogues were inhibitors. Similarly, amiloride did not alter Ca2+ release in the presence or absence of Na+. However, some analogues were found that stimulated and others that inhibited Ca2+ release. While many of these analogues reduced mitochondrial respiratory control ratios, two analogues were identified which inhibited Ca2+ uptake but did not alter mitochondrial respiratory control. Similarly two analogues were identified which inhibited Ca2+ efflux without affecting respiratory control.     

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.     

A comparison of phlorizin and phloretin adsorption by the tapeworm Hymenolepis diminuta

Phloretin and phlorizin adsorb to the tegument surface of Hymenolepis diminuta, with KDs of 2.39 mM and 14.7 microM, respectively, and Vmaxs of 1446 and 12.54 nmoles/g tissue per 2 min, respectively. Phloretin adsorption is not inhibited by phlorizin or glucose. Glucose partially inhibits phlorizin adsorption. Phlorizin, but not phloretin, adsorption to isolated tegument brush border membrane preparations is partially inhibited by N-ethylmaleimide. No indications of phlorizin hydrolysis to phloretin during incubation with H. diminuta were obtained. The data are supportive of spacially separate and distinct binding sites for phloretin and phlorizin in the tegument brush border.     

Lack of effect of charge of phlorizin and phloretin on mutarotase action.

Many transport phenomena are inhibited by the polyphenolic compounds phlorizin and its aglucone phloretin. We report here a hitherto unsuspected charge on the molecules at physiological pHs which may be of importance in interpreting their mode of action. The change in inhibition of mutarotase was studied with change of pH with these compounds, but no significant effect of charge was detected.     

The effects of phlorizin,phloretin and ouabain on the reabsorption of glucose by the Malpighian tubules of Locusta migratoria migratorioides

Isolated Malpighian tubules of Locusta reabsorb significant levels of glucose from their lumen back into the bathing fluid (haemolymph). This reabsorption is inhibited by phlorizin, phloretin and ouabain. Both phlorizin and phloretin are found to accumulate in the secreted fluid of the tubules against concentration gradients. Ouabain inhibition is explained in terms of its effect on intracellular Na⁺ concentrations. A hypothetical model of the role that Na⁺ may play in glucose reabsorption is presented as a possible explanation of these observations.     

Inhibition of glucose transport in human erythrocytes by benzylalcohol.

The inhibition of glucose transport by the non-ionizable local-anesthetic denzylalcohol is of the mixed type and independent of pH. The affinity of benzylalcohol to the free carrier is about three times larger than that to the carrier-glucose complex.     

Inhibition of nucleoside transport by new analogues of nitrobenzylthioinosine

Nitrobenzylthioinosine (NBTI, 1) was systematically modified by attachment of substituents at positions C6 and N9, and also by substitution of N1 with C. These modifications were chosen to reduce the polarity of the new compounds. Incorporation of the nitro functionality into a benzoxadiazole ring system was considered first. These new nucleosides showed high affinity (1.5-10nM) towards the nucleoside transport protein as present on human erythrocyte ghosts. Next, modification of this benzoxadiazole ring system with C, S and O in different positions produced a number of less polar nucleosides with affinity in the higher nanomolar range. Modification of N9 was achieved with different alkyl and alcohol substituents. An n-butyl substituent proved best, although all variations yielded substantial decreases in affinity. Replacement of N1 by a carbon atom in combination with a 2-Cl substituent also resulted in a relatively potent NBTI derivative (47 nM).     

Probing the antioxidant potential of phloretin and phlorizin through a computational investigation

The structures and energetics of two dihydrochalcones (phloretin and its glycoside phlorizin) were examined with density functional theory, using the B3LYP, M06-2X, and LC-ω PBE functionals with both the 6-311G(d,p) and 6-311 + G(d,p) basis sets. Properties connected to antioxidant activity, i.e., bond dissociation enthalpies (BDEs) for OH groups and ionization potentials (IPs), were computed in a variety of environments including the gas-phase, n-hexane, ethanol, methanol, and water. The smallest BDEs among the four OH groups for phloretin (three for phlorizin) were determined (using B3LYP/6-311 + G(d,p) in water) to be 79.36 kcal/mol for phloretin and 79.98 kcal/mol for phlorizin while the IPs (at the same level of theory) were obtained as 139.48 and 138.98 kcal/mol, respectively. By comparing with known antioxidants, these values for the BDEs indicate both phloretin and phlorizin show promise for antioxidant activity. In addition, the presence of the sugar moiety has a moderate (0-6 kcal/mol depending on functional) effect on the BDEs for all OH groups. Interestingly, the BDEs suggest that (depending on the functional chosen) the sugar moiety can lead to an increase, decrease, or no change in the antioxidant activity. Therefore, further experimental tests are encouraged to understand the substituent effect on the BDEs for phloretin and to help determine the most appropriate functional to probe BDEs for dihydrochalcones.     

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.