Donald F. Diedrich

Competitive inhibition of intestinal glucose transport by phlorizin analogs

Abstract The nature of the inhibition of intestinal glucose transport in vitro by phlorizin and its congeners was investigated. The results of two series of experiments are described. In one, the rate of transport was measured as glucose lost from the medium after 10 minutes of incubation with hamster intestine, and in the other, the amount of glucose accumulated in the tissue was taken as a measure of transport rate. In both sets of experiments, glucose in the medium was varied over a 10-fold concentration range while dosage of a particular inhibitor was held constant. The kinetic data obtained from these experiments were analyzed graphically as double reciprocal plots, and are interpreted as evidence that phlorizin and its glycosidic analogs compete with glucose for the transport carrier. On the other hand, the results of the experiments with phloretin indicate that this aglycone inhibits transport in a non-competitive manner. The apparent dissociation constants of the inhibitors were determined, and the relative free energies of interaction were calculated and compared. These results provide a basis for further work in which the development of an in vitro assay for the glucose carrier in cell-free homogenates could be attempted.

Is phloretin the sugar transport inhibitor in intestine

Abstract Isolated brush border fragments from hamster small intestine epithelium were incubated with tritiated phlorizin and direct measurements of the interaction were made. The adsorption kinetics were found to be unexpectedly complex involving at least two reactions: An instantaneous adsorption, representing the interaction of phlorizin with a readily accessible receptor site, is followed by a much slower secondary uptake of radioactivity by the tissue fragments. The first reaction can be dissected from the other process; it exhibits first order kinetics and fits a Langmuir adsorption isotherm. The dissociation constant for this interaction has a maximum value of 75 μ m . An estimate of the receptor density on the epithelial cell was also obtained from these data. The brush border membrane of each cell possesses from 2 to 4 × 10 8 phlorizin receptor sites. The relationship of this estimate to the number of sugar carrier molecules per cell is discussed. The secondary uptake of radioactivity by the brush border was attributable to the adsorption of [ 3 H]phloretin which was enzymatically generated by a β-glucosidase present in the brush border membrane. The implications of this conversion of phlorizin to the aglycone, under common incubation conditions are discussed and the question is raised whether phloretin, rather than the parent glucoside, is the actual inhibitor of the carrier transport mechanism in the intestine. The preparation of tritiated phlorizin is described and the methods which can be used to radiochemically purify the compound are presented.

Inhibition of [3H]phlorizin binding to isolated kidney brush border membranes by phlorizin-like compounds☆

Abstract 1. 1. Earlier work has demonstrated that (a) isolated brush border membranes from rat kidney cortex possess a high-affinity receptor for phlorizin; binding is dependent upon the presence of sodium and is competitively blocked by d -glucose, (b) phlorizin competitively inhibits active transport of glucose across rat kidney proximal tubules and (c) a series of phlorizin-like compounds possess a characteristic, wide range of inhibitory potency as glucose transport poisons in kidney and intestinal systems. 2. 2. 3H-labeled phlorizin binding to the brush border membrane site has now been shown to be inhibited by this series of phlorizin analogs, and the critical finding is that the previosuly established relative potencies of these compounds as transport inhibitors parallel their ability to block phlorizin binding to the specific membrane receptor. 3. 3. These results increase the weight of evidence for the view that the glucose-transport system is retained as a structurally unaltered component of these isolated brush border fragments. 4. 4. A view of the conformation, which the phlorizin receptor could assume in the matrix of the outer membrane, is presented in the discussion.

Photoaffinity labeling and identification of (a component of) the small-intestinal Na+, D-glucose transporter using 4-azidophlorizin

Unlabeled phlorizin was purchased from Carl Roth, Karlsruhe (FRG). Low levels of phloretin in the commercial product were removed by silica gel column chromatography, using methylene chloride-methanol, 4: 1 (v/v) as the eluent, and the glucoside was recrystallized from warm water. The synthesis of paraphlorizin (phloretin 4’-glucoside; fig.1, III) has been described before [2]. The photoreactive 4-azidophlorizin and its ‘H-labelled analogue were synthesized and chemically characterized as described elsewhere [I]. 4-[3H]Azidophlorizin had a specific activity of 0.405 Ci mmol-’ and according to the tritiatlon procedure

Electron spin resonance investigation of the interaction of the anion and glucose transport inhibitor, p-azidobenzylphlorizin, with the human red cell membrane

The membrane perturbations caused by the interaction of p-azidobenzylphlorizin (p-AzBPhz), a potential photoaffinity labeling agent of the anion and D-glucose transporters in the human erythrocyte, have been studied using electron spin resonance (ESR) spectrometry. Two lipid-specific spin labels have been employed; one of these agents, a hexadecyl-quarternary amine with the nitroxide reporter group covalently attached to the cationic nitrogen, (CAT-16), has been used to monitor changes in the physical state of the membranes extracellular phospholipid/water interface. The other spin label, 5-doxylstearic acid (5-NS), is designed to examine the order and motion of the lipid bilayer near the cell surface. In separate experiments, intact human red cells labeled with these lipid-specific spin labels were exposed to small amounts of the phlorizin azide. A dose-dependent alteration in CAT-16 motion was observed, but the p-AzBPhz interaction with the membrane had no effect on the spectrum of 5-NS. The half-maximal effect of the phlorizin derivative on the CAT-16 spectrum occurred when about 2 million molecules were bound to each cell. This is also the combined amount of band 3 and band 4.5 present in the red cell membrane and represents the concentration necessary to inhibit both anion and glucose transport. Our results suggest that the first p-AzBPhz molecules binding to the red cell membrane interact with the anion and sugar transporters, and not with the bulk lipid bilayer.

The kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border β-glucosidase

Abstract Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the β-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 by 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.

Active site comparison of mutarotase with the glucose carrier in human erythrocytes

Abstract The relative effectiveness of a number of diphenols related in structure to phloretin as reversible inhibitors of mutarotase was compared to their known potencies as competitive glucose carrier poisons in human erythrocytes. A remarkable parallelism was found, and the results are discussed with a view of encouraging further investigations designed to determine whether this socalled mutarotase protein and the ubiquitous sugar carrier are one and the same molecule. More than 30 phenolic compounds have been used as molecular probes to partially elucidate the topography of of the active center of mutarotase.

A hydrolase-related transport system is not required to explain the intestinal uptake of glucose liberated from phlorizin

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.

Kinetic studies of D-glucose transport in renal brush-border membrane vesicles of streptozotocin-induced diabetic rats

Renal brush-border membrane vesicles prepared from streptozotocin-induced 4-day-diabetic rats possessed a Na+-dependent D-glucose transport system that exhibited apparent Kt and Vmax values about 2-fold greater than normal. Apparently, hyperglycemia and probably other stimuli cause the induction and membrane incorporation of a low-affinity transporter in these membranes; this increased sugar-transport capacity is retained for at least 4 weeks so long as the animals maintained or increased their body weight. Membranes prepared from 28-day-diabetic, severely ill ketoacidotic animals lose this enhanced transport ability and the decrease in Vmax was found to correlate directly with the weight loss. Furthermore, the transporter in brush-border membranes prepared from these cachectic animals had an even lower affinity for glucose than those from the acute hyperglycemic animals. That these changes in the diabetic animals represent major alterations in renal brush-border membrane construction is further supported by our observation that the specific activity of the marker enzymes, alkaline phosphatase and neutral alpha-glucosidase, are profoundly increased and decreased, respectively, in this condition.

The affinity of phlorizin-like compounds for a β-glucosidase in intestinal brush borders: Comparison with the glucose transport system☆

Abstract The ability of a series of phlorizin-like glycosides to interact with the active center of the β-glucosidase (phlorizin hydrolase) present in hamster intestinal brush border membranes has been estimated from K m and K i measurements. All of the glycosides except para -phlorizin, were cleaved at the same V although large differences in their apparent K m values were found. The inhibitors of [ 3 H]glucose-labeled phlorizin hydrolysis all acted competitively and their K i values equaled their respective K m s with two exceptions: (1) para -phlorizin, an isomer with its sugar residue in a different orientation than any of the other derivatives, is a better inhibitor than substrate and (2) phloretin 2′-galactoside appeared to be a better substrate than inhibitor: it was extensively hydrolyzed, more than would have been predicted from its apparent K i value. This “extra” hydrolysis was probably due to lactase action. The affinities of these compounds for the glucose transport system and phlorizin hydrolase were compared. The relative inhibitory potency of all but two of the analogs was the same in either system suggesting that there are similarities in the architecture of the two receptors. However, there are differences which indicate that these two membrane components do not share a common site: (1) The K i values of the glycosides as sugar carrier blockers are 100-fold smaller than their K i S as hydrolase inhibitors, (2) 4-methoxyphlorizin has somewhat greater affinity for the β-glucosidase than the transporter, and (3) para -phlorizin has essentially no affinity for the glucose carrier but is a good substrate and potent inhibitor of phlorizin hydrolase. Studies designed to isolate and affinity label the phlorizin-receptor component of the transport system must take into account that the intestinal brush border membrane possesses a β-glycosidase whose active site will probably also be labeled and at which phlorizin-like ligands could be inactivated.