D-glucose Uptake Research Articles

Nutrient uptake by rat enterocytes during diabetes mellitus; evidence for an increased sodium electrochemical gradient.

1. The effect of streptozotocin-induced diabetes (7 day duration) in rats on D-glucose uptake in vivo, the unidirectional uptake of D-glucose and L-proline in vitro, the passive uptake of L-glucose in vitro and the potential difference across the brush-border membrane has been studied. 2. Diabetes resulted in an increased carrier-mediated glucose uptake both in vivo and in vitro and a stimulation of L-proline uptake at a concentration of the amino acid (0.025 mM) at which uptake was largely Na+ dependent. Diabetes was without effect on uptake using a proline concentration of 50 mM at which transport was predominantly Na+ independent. 3. A marked hyperpolarization of the brush-border membrane and an enhanced passive glucose uptake were also evident during diabetes. 4. We conclude that the stimulation of glucose uptake in vivo in diabetic intestine involves events at the brush-border membrane. The mechanisms include an increased surface area for uptake and an enhanced transmembrane electrical gradient. The latter will have a major effect on the transport of other substrates when the uptake pathway is primarily Na+ dependent.

Glucagon effect on myocardial transport and utilization of energy-metabolites from the coronary microcirculation in the perfused rabbit heart

Effect of glucagon on energy-metabolite transport into cardiac muscle was studied during a single transit through the isolated rabbit heart using a rapid paired-tracer dilution method. Kinetic experiments revealed that 1.5 microM glucagon stimulated the influx of palmitate bound to 30 g/litre albumin, by increasing the V 2.3 times and increasing the Km for transport 2.4 times. Tracer uptake of D-glucose, as the only exogenous substrate provided, was increased by 80% by 1.5 microM glucagon. Myocardial utilization of [3H]-or [14C]-labelled short-chain monocarboxylic acids (L-lactate, pyruvate and acetate) was significantly reduced by glucagon, to the same degree as their unidirectional sarcolemmal transport. Inhibition of L-[14C]lactate uptake was dose-dependent and in positive correlation with myocardial lactate production. It is concluded that glucagon may regulate sarcolemmal permeability and myocardial utilization for energy-metabolites from the coronary circulation.

The action of phenylarsine oxide on the stereospecific uptake of D-glucose in basal and insulin-stimulated rat adipocytes

Phenylarsine oxide (PAO) has been used to inhibit the stereospecific uptake of D-glucose in basal and insulin-stimulated rat adipocytes. The inhibition is dose dependent and is partially reversed by dithiothreitol. The results are consistent with a direct interaction between the glucose transporter and PAO. By manipulating the sequence of exposure of cells to PAO and insulin it is possible to differentiate between the effects of PAO on transport into cells with receptor-rich and transporter-rich plasma membranes. PAO rapidly inhibits transport in insulin-stimulated adipocytes but at low concentrations inhibition is transient and recovery of stereospecific uptake takes place after approx. 20 min. The results can be interpreted in terms of the recruitment mechanism of insulin stimulation of transport and demonstrate that a relatively large intracellular pool of transporters exists after insulin stimulation. It also follows that sulphydryl groups probably play a critical role in the mechanism of glucose uptake.

Transport and metabolism of d-glucose in human adipocytes. Studies of the dependence on medium glucose and insulin concentrations

Uptake and metabolism of the physiologically labelled d-glucose ( d-[U- 14C]glucose) has been characterized in human adipocytes at several unlabelled d-glucose concentrations in the absence and presence of insulin. Following a 90 min incubation, about 80% of the intracellular radioactivity was incorporated into total lipids at tracer glucose concentration, as well as at higher glucose concentrations in basal and insulin-stimulated cells, whereas 20% was recovered as hydrophilic metabolites. The only 14C-labelled metabolite escaping the cells in detectable amounts was CO 2, which accounted about 4%. At trace glucose concentrations (5 μmol/l), the rate of glucose uptake was linear with time. Comparative studies of initial glucose uptake after 10 s and tracer d-glucose conversion to total lipids after 90 min showed high coefficients of correlation between basal rates ( r = 0.87), maximal response above basal level to insulin ( r = 0.92) and insulin sensitivity ( r = 0.78). Thus, under these conditions glucose transport is rate-limiting for net glucose uptake, and measurements over long time intervals of rates for total cell-associated radioactivity or lipogenesis may serve as reliable estimates of initial glucose influx rates. However, the conversion rate of tracer glucose to metabolites decreased progressively with the glucose concentration and with an apparent K m of about 0.2 mmol/l. The three metabolic pathways exhibited similar percentage decreases in their activities, suggesting that a common enzymatic step is rate-limiting. In comparison, the K m for initial d-glucose uptake rate was about 7 mmol/l. Hence, the capacity for total glucose metabolism comprised only a small fraction of the glucose transport capacity at medium glucose concentrations above tracer concentrations. Both basal, half-maximal and maximal insulin-stimulated rates of adipocyte glucose utilization were dependent on the glucose concentration. Thus, comparing lipogenesis at tracer and at 0.5 mmol/l medium glucose concentration, it was shown that the higher medium glucose concentration was associated with a 60% lowering of the basal rate, a 35% reduction in the percentage response above baseline to maximal insulin stimulation and a 4-fold increase in the insulin sensitivity. Obviously, these findings reflect some intracellular step(s) being rate-limiting at medium glucose levels above tracer values.

Changes in glucose uptake by and phlorizin binding to brush-border membrane vesicles of small intestine from streptozotocin-induced diabetic rats.

The changes in the intestinal glucose transport in streptozotocin-induced diabetic rats have been demonstrated with brush-border membrane vesicles. When Na+-dependent D-glucose uptake and phlorizin binding activities were compared, both significantly increased either in the jejunum or ileum of diabetic rats compared with those of control. Kinetic studies showed the increases in the Vmax of glucose transport and the maximum binding of phlorizin (Bmax), whereas the Kt and Kd remained unchanged, respectively. These results suggested that the increase in glucose transport in diabetic rat intestine was due to the increase in the number of the intact glucose transporters.

Genetic and phenotypic adaptation of intestinal nutrient transport to diet in fish.

1. Herbivores have higher rates of intestinal sugar transport and lower rates of amino acid transport than carnivores, if each are studied while eating their respective natural diets. It was unclear whether these species differences involve a genetic contribution, since when omnivores are switched from a high-protein to a high-carbohydrate diet they reversibly increase sugar transport and suppress amino acid transport. Hence we studied eight fish species of differing natural diets while all were eating the same manufactured diet. 2. Na+-dependent L-proline uptake and active D-glucose uptake, measured in vitro by the everted intestinal sleeve technique, followed Michaelis-Menten kinetics. Values of the apparent Michaelis-Menten constant increased with values of the maximal transport rate, probably as a result of unstirred layer effects. 3. The ratio of proline to glucose uptake decreased in the sequence: carnivores greater than omnivores greater than herbivores. The intestine's uptake capacity for the non-essential nutrient glucose was much higher in herbivores than in carnivores, correlated with species differences in carbohydrate content of the natural diet. Proline uptake varied much less among species, since species with different natural diets still have similar protein requirements. 4. Since all species were studied while eating the same diet, these species differences in uptake are not phenotypic but genetic adaptations to the different natural diets. 5. In two fish species which normally switch from carnivory towards herbivory or omnivory as they mature, we observe a 'hard-wired' developmental change in intestinal uptake. Larger animals had lower proline uptake relative to glucose uptake than did smaller animals, even though both were being maintained on the same diet in the laboratory. 6. Carnivorous fish tend to allocate absorptive tissue to pyloric caeca or a thick mucosa, while herbivorous fish tend towards a long thin intestine.

Chelation of Intracellular Calcium Prevents Stimulation of Glucose Transport by Insulin and Insulinomimetic Agents in the Adipocyte. Evidence for a Common Mechanism*

Vanadate, concanavalin A (Con A), H2O2, and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), seemingly unrelated compounds, have effects on cellular metabolism similar to those of insulin. The mechanism(s) by which these diverse compounds act and their relevance to insulin action remain unclear. The hypothesis that increased intracellular calcium ([Ca2+]i) may provide the common basis for the stimulatory effects of these agents on glucose uptake in adipocytes was tested. This was accomplished by preincubating cells with the membrane-permeant ester, quin2-AM (2-[(2-bis[carboxymethyl] amino-5-methylphenoxy]-6-methoxy-8-bis[carboxymethyl]- aminoquinolinetetrakis [acetoxymethyl] ester), which is rapidly accumulated and hydrolyzed by esterases to form the impermeant tetracarboxylate chelator form, quin2. Vanadate, Con A, H2O2, and TPA stimulate D-glucose uptake in adipocytes approximately 3-, 3-, 2-, and 0.6-fold, respectively, compared to the 5- to 10-fold stimulation of D-glucose uptake obtained with insulin. Preincubation with quin2-AM produced a dose-dependent inhibitory effect only on the stimulated portion of glucose transport without affecting basal (unstimulated) transport. The concentrations for half-maximal inhibition (IC50) of stimulated glucose transport by quin2-AM were 26, 35, 25, 14, and 34 microM for insulin, vanadate, Con A, H2O2, and TPA, respectively. Quin2-AM maximally inhibited stimulated glucose uptake by greater than 85% for all of the insulin mimetic agents. In contrast the maximal inhibition of insulin-stimulated glucose transport by quin2-AM was 55 +/- 4%. Therefore, stimulation of glucose transport by insulin and other diverse compounds appears to involve at least one common calcium-dependent intermediate step.

The Na+-independent D-glucose transporter in the enterocyte basolateral membrane: orientation and cytochalasin B binding characteristics.

Phloridzin-insensitive, Na+-independent D-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed a Kt of 48 mM and a Vmax of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lower Kt of 23 mM and a Vmax of 0.20 nmol glucose/mg protein/sec. D-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. The Ki for cytochalasin B as an inhibitor of glucose transport was 0.11 microM and the KD for binding to the carrier was 0.08 microM. D-glucose-sensitive sensitive binding of cytochalasin B to the membrane preparation was maximized with L- and D-glucose concentrations of 1.25 M. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na+-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and the Kt for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.

Formula omitted] as a competitive inhibitor of Na +-dependent d-glucose cotransporter in the small intestine brush-border membrane

1-O-n- Octyl-β- d-glucopyranoside is a competitive inhibitor of the Na +-dependent d-glucose uptake into rabbit, rat and human intestinal brush-border membrane vesicles. The lack of effect on the equilibrium uptake demonstrates that the detergent does not act by rupturing the vesicles; no membrane leakiness was apparent at the concentrations of octylglucopyranoside used, since d-glucose uptake is not inhibited even in the absence of the Na + gradient (in K + solution). There is a competitive interaction between octylglucopyranoside and d-glucose, as shown by Dixon and by Hunter and Down plots. The selectivity of the detergent effect is confirmed by its modest influence on amino acid uptake.

Inhibition of alkaline phosphatase activity and d-glucose uptake in rat renal brush-border membrane vesicles by aminoglycosides

The binding of aminoglycoside antibiotics to, and their effects on, the plasma membrane were studied using isolated rat renal brush-border membrane vesicles. Dibekacin was noted to bind with brush-border membrane vesicles having a single class of many binding sites. 3H-labeled dibekacin binding was inhibited competitively by unlabeled dibekacin, gentamicin or amikacin. The inhibition constants obtained from the Dixon plots followed the order of gentamicin ≅ dibekacin > amikacin. The alkaline phosphatase activity of brush-border membrane vesicles was inhibited by gentamicin significantly, as was also observed by a histochemical study. Sodium-dependent d-glucose uptake by brush-border membrane vesicles was significantly inhibited by the addition of gentamicin.

Ischemia induces surface membrane dysfunction. Mechanism of altered Na+-dependent glucose transport.

Reversible ischemia reduced renal cortical brush border membrane (BBM) Na+-dependent D-glucose uptake (336 +/- 31 vs. 138 +/- 30 pmol/mg per 2 s, P less than 0.01) but had no effect on Na+-independent glucose or Na+-dependent L-alanine uptake. The effect on D-glucose uptake was present after only 15 min of ischemia and was due to a reduction in maximum velocity (1913 +/- 251 vs. 999 +/- 130 pmol/mg per 2 s; P less than 0.01). This reduction was not due to more rapid dissipation of the Na+ gradient, altered sidedness of the vesicles, or an alteration in membrane potential. Ischemia did, however, reduce the BBM sphingomyelin-to-phosphatidylcholine (SPH/PC) and cholesterol-to-phospholipid ratios and the number of specific high-affinity Na+-dependent phlorizin binding sites (390 +/- 43 vs. 146 +/- 24 pmol/mg; P less than 0.01) without altering the binding dissociation constant (Kd). 20 mM benzyl alcohol also reduced the number of Na+-dependent phlorizin binding sites (418 +/- 65 vs. 117 +/- 46; P less than 0.01) without altering Kd. The reduction in Na+-dependent D-glucose transport correlated with ischemic-induced changes in the BBM SPH/PC and cholesterol-to-phospholipid ratios and membrane fluidity. Taken together these data indicate the cellular site responsible for ischemic-induced reduction in renal cortical transcellular glucose transport is the BBM. We propose the mechanism involves marked alterations in BBM lipids leading to large increases in BBM fluidity which reduces the binding capacity of Na+-dependent glucose carriers. These data indicate that reversible ischemia has profound effects on the surface membrane function of epithelial cells.

Proximo-distal gradient of Na +-dependent D-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine

Brush-border membrane vesicles were isolated from the jejunum and ileum of 17–20-week-old normal human fetuses and found to be highly enriched in sucrase activity with less than 5% contamination by basolateral membranes. Time course studies of D-glucose uptake clearly showed a transient, phlorizin-sensitive, and Na +-dependent accumulation of sugar into these vesicles. Higher maximum overshoot values and initial rates of D-glucose uptake were recorded in jejunal as compared to ileal vesicles while low substrate binding to the membranes, identical intravesicular volumes and equivalent dissipation of the Na +-gradient were found in the two preparations. It was concluded that a fully functional Na +-D-glucose cotransport system is present with a proximo-distal gradient of activity during the early gestation period.

Direct effects of biguanides on glucose utilization in vitro

The effect of the biguanides metformin and phenformin on glucose utilization in isolated cells was studied with IM-9 human lymphocytes. Both agents stimulated glucose consumption from the incubation media. Detectable effects of metformin were seen at 33 μmol/L and detectable effects of phenformin were seen at 1.7 μmol/L. Both agents, at similar concentrations, also stimulated [ 3H] 2-deoxy- d-glucose uptake. Studies with phenformin indicated that biguanides increase the V max of uptake without changing the Km. In contrast to the biguanides, IM-9 cells insulin did not influence either glucose consumption or [ 3H] 2-deoxy- d-glucose uptake. These data provide evidence, therefore, that biguanides may directly influence the cellular utilization of glucose.

Decreased transport of d-glucose and l-alanine across brush-border membrane vesicles from small intestine of rats treated with mitomycin C

To elucidate the mechanisms underlying the dysfunctions of intestinal absorption induced by antitumor drugs, the effect of pretreatment with mitomycin C on sodium gradient-dependent d-glucose and l-alanine transports was studied in rat brush-border membrane vesicles. 24, 48, 96, or 120 h following a single intravenous injection of mitomycin C, brush-border membrane vesicles were prepared from rat small-intestines. The uptake of d-glucose and l-alanine was shown to be Na + gradient-dependent even in the case of vesicles obtained from mitomycin C-treated rats, but uptake rates measured at 15 s and magnitude of overshooting effect in uptake of both solutes were decreased in vesicles maximally from 48 h mitomycin C-treated rats. The rate of d-glucose uptake calculated at 15 s recovered to the control level in vesicles prepared at 96 h and 120 h after mitomycin C-treatment, indicating that the effect of mitomycin C on Na + gradient-dependent d-glucose transport would be fully reversible. Tracer exchange experiments under Na + and d-glucose equilibrated conditions indicated that the Na +/ d-glucose transporters were similarly operative in the vesicles from control and 48 h mitomycin C-treated rats. Rates of 22Na + uptake measured at 15 s in vesicles from 48 h mitomycin C-treated rats, however, were increased. The increased permeability to Na + might bring about a more rapid dissipation of the Na + gradient in these vesicles and this would secondarily cause the decrease in Na +-dependent d-glucose uptake in vesicles from mitomycin C-treated rats.

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, l-glutamate, l-proline, l-alanine, and α-methyl-glucopyranoside (α-methyl d-glucoside) were accumulated through Na +-dependent and Na +-independent transport pathways. d-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na +. Influx of inorganic phosphate was only observed in the presence of Na +. Na +/α-methyl d-glucoside uptake was preferentially inhibited by phlorizin and d-glucose uptake by cytochalasin B. An amiloride-sensitive Na +-transport was also identified. In isolated apical vesicles (enriched 8-fold in γ-glutamyltransferase), l-glutamate, l-proline, l-alanine, α-methyl d-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na +-gradient as compared to an inwardly directed K +-gradient. l-Glutamate transport required additionally intravesicular K +. d-Glucose transport was similar in the presence of a Na +- and a K +-gradient. Na +/α-methyl d-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na +/ d-glucose transport. An amiloride-sensitive Na +/H + exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na +-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. d-Glucose appears a poor substrate for the Na +/hexose transport system.

Identification of D-glucose-binding polypeptides which are components of the renal Na+-D-glucose cotransporter.

D-Glucose-binding polypeptides in the Na+-D-glucose cotransporter from pig renal cortex were identified by affinity labeling with two D-glucose analogs, 10-N-(N-[4-azido-2-nitrophenyl]-beta-alanyl)amino-1-decyl-beta-D- glucopyranoside (NapADG) and 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside (BADG). During short-term incubation in the dark, NapADG and BADG are reversible inhibitors of Na+ gradient-dependent D-glucose uptake and Na+-dependent phlorizin binding with Ki values of about 40 and 400 microM, respectively. Irreversible inhibition of Na+-dependent phlorizin binding, which was prevented by D-glucose or phlorizin, was measured after a 1-h incubation with BADG. Both NapADG and BADG selectively labeled polypeptides with apparent molecular weights of 82,000, 75,000, 64,000, and 47,000. Since labeling of the Mr 82,000 and 75,000 polypeptides by both analogs was partially dependent on the presence of Na+ and was partially protected by D-glucose or phlorizin but not by L-glucose or D-mannose, these polypeptides are thought to be components of the renal Na+-D-glucose cotransporter which contain D-glucose-binding sites. For the Mr 64,000 and 47,000 polypeptides, Na+ dependence and D-glucose protection were not constantly observed. However, also, these polypeptides are thought to be components or proteolytic splitting products of the Na+-D-glucose cotransporter since we observed that three monoclonal antibodies showed cross-reaction with the BADG-labeled Mr 82,000, 64,000, and 47,000 polypeptides (K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, and H. Koepsell, manuscript in preparation). When the BADG-labeled Mr 82,000 and 75,000 polypeptides were analyzed after two-dimensional separation by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, three-labeled, D-glucose-protectable polypeptides with the respective molecular weights and isoelectric points of 82,000 and 5.6, 75,000 and 5.4, and 75,000 and 6.9 were distinguished. The data indicate that renal brush-border membranes contain several polypeptides which are components of the Na+-D-glucose cotransporter and contain D-glucose-binding sites.

16. DEMONSTRATTCN OF DEFECTIVE JEJUNAL BRUSH BORDER Na+-COUPLED GLUCOSE TRANSPORT IN CONGENITAL GLUCOSE-GALACTCSE MALABSORPTION

Studies in congenital glucose-galactose malabsorption (GGM) have indicated an abnormality in small intestinal glucose and galactose transport but the precise site of the defect has not been defined. We have therefore investigated Na+-coupled D-glucose uptake, and Na+/H+ exchange in a patient with GGM using brush border membrane vesicles (BBMV) prepared from jejunal biopsies using a miniaturised Mg++-precipitation technique. The diagnosis was confirmed by clinical presentation, response to diet and jejunal perfusion studies, which showed markedly defective glucose and galactose absorption (p < 0.001) and normal fructose transport.

Evidence for a high-affinity sodium-dependent D-glucose transport system in the kidney

Renal brush-border membrane D-glucose transport was studied in vesicles prepared from early proximal tubules (outer cortical tissue) and late proximal tubules (outer medullary tissue). Kinetics of sodium-dependent D-glucose uptake, determined with zero trans sodium gradient of 200 mM at 21 degrees C demonstrated a curvilinear Woolf-Augustinsson-Hofstee plot in both cortical and medullary vesicles. Three sodium-dependent systems were discerned in the cortex, possessing Km values of 12.35 +/- 2.2 mM, 520 +/- 39 microM, and 12 +/- 1 microM, respectively. In contrast, only the high-affinity, low-capacity systems were present in the medullary tissue; with respective Km values of 484 +/- 95 microM and 11 +/- 1 microM and mean Vmax values of 2.4 +/- 0.1 and 0.44 +/- 0.08 nmol X mg protein-1 X min-1. The high-affinity systems were more abundant in the medulla than in the cortex. These studies support the notion of multiple transport processes for sodium-dependent D-glucose transport in the pig kidney. Further, they provide evidence for a third, high-affinity system located principally in the medullary tissue, perhaps the late proximal tubule.

Insulin stimulation of adipocyte membrane glucose transport: A graded biologic response insensitive to bilayer lipid disordering

Aspects of the mechanism by which insulin stimulates the membrane glucose transport system were examined by (1) assessing the influence of the bilayer lipid structure on transport stimulation characteristics, and (2) considering the form of the insulin dose-response curve. We tested the effects of membrane lipid perturbation on the insulin stimulation process. Benzyl alcohol, at concentrations (25 mM) that grossly fluidize lipids forming the adipocyte membrane bilayer matrix, caused 50% inhibition of intrinsic transporter activity. However, this membrane perturbation had no significant effect on either the insulin dose-response curve (conducted at 37°) or the time-course of the insulin stimulation of hexose transport (conducted at 32°). These data are difficult to rationalize in terms of a model in which transport stimulation involves interaction of transporters and hormone-bound receptors that is limited by lateral diffusion of these proteins in the fluid lipid bilayer. Curve-fitting experimental insulin dose-response data for stimulation of 2-deoxy- d-glucose and d-glucose uptake provided an estimate of an insulin “association constant” for transport regulation that may be compared with recent insulin receptor binding data. Similar magnitude constants were obtained whether estimated directly from plots of transport velocity versus arithmetic hormone dose, or by extrapolation from linear segments of sigmoidal velocity versus log dose plots, or from inverse (Lineweaver-Burk-type) plots of the insulin dose-response data. Insulin apparently regulates transport by associating with a binding site, having an apparent dissociation constant which is determinable through kinetic measurements of hexose uptake ( K D app approx. 17−40 pM). This is in good agreement with the dissociation constant, K D , determined from Scatchard plots of recent binding data to adipocytes, for a class of receptors representing the “high affinity” binding sites for insulin. Insulin dose-response curve simulations also indicated that the stimulation process may be classified in pharmacologie terms as a typical graded biologic response and may involve insulin association with a site that regulates transport rates in a manner kinetically analogous to allosteric modulation of a V-series enzyme by a noncompetitive ligand. From the results we suggest that (1) a relatively close association occurs between transport and receptor proteins in the membrane, where the relative activation of transport depends on the fractional occupancy of functional high affinity receptors by insulin, and (2) the insulin stimulation of transport involves regions of the membrane that are not influenced significantly by disordering the membrane lipid matrix.

Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles

High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2,000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control; with both techniques, the concentration of dextran after being taken up into the vesicles was similar to that in the incubation medium, suggesting attainment of equilibrium. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.