cytochalasin-b and Hyperglycemia

Individuals with non-insulin dependent or insulin-dependent diabetes mellitus present insulin resistance in peripheral tissues. This is reflected in a subnormal whole body insulin-dependent glucose utilization, largely dependent on skeletal muscle. Glucose transport across the cell membrane of this tissue is rate limiting in the utilization of the hexose. Therefore, it is possible that a defect exists in insulin-dependent glucose transport in skeletal muscle in diabetic states. This review focuses on two questions: is there a defect at the level of glucose transporters in skeletal muscle of diabetic animal models, and is this a consequence of abnormal insulin or glucose levels? The latter question arises from the fact that these parameters usually vary inversely to each other. Glucose transport into skeletal muscle occurs by two membrane proteins, the GLUT1 and GLUT4 gene products. By subcellular fractionation and Western blotting with isoform-specific antibodies, it was determined that isolated plasma membranes (PM) contain GLUT4 and GLUT1 proteins at a molar ratio of 3.5:1 and that an intracellular fraction (internal membranes; IM) different from sarcoplasmic reticulum contains only GLUT4 transporters. The IM furnishes transporters to the PM in response to insulin. Both transporter isoforms bind cytochalasin B in a D-glucose-protectable fashion. In streptozocin-induced diabetes of the rat with normal fasting insulin levels and marked hyperglycemia, the number of cytochalasin B-binding sites and of GLUT4 proteins diminishes in the PM whereas the GLUT1 proteins increase to a new ratio of about 1.5:1 GLUT4:GLUT1. In the IM, the levels of GLUT4 protein drop, as does the cellular GLUT4 mRNA. To investigate if these changes are associated with hyperglycemia, glucose levels were corrected back to normal values for a 24-h period with sc injections of phlorizin to block proximal tubule glucose reabsorption. This treatment restored cytochalasin B binding, restored GLUT4 and GLUT1 values back to normal levels in the PM, and partly restored cytochalasin B binding but not GLUT4 levels in the IM, consistent with only a partial recovery of GLUT4 mRNA. It is concluded that GLUT4 protein in the PM correlates inversely whereas GLUT1 protein correlates directly with glycemia. It is proposed that the decrease in GLUT4 levels is a protective mechanism, sparing skeletal muscle from gaining glucose and experiencing diabetic complications, albeit at the expense of becoming ins

Topics: Animals; Binding, Competitive; Blood Glucose; Cytochalasin B; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Gene Expression Regulation; Glucose Transporter Type 4; Glycosylation; Humans; Hyperglycemia; Insulin; Insulin Resistance; Intestinal Absorption; Kidney Tubules, Proximal; Monosaccharide Transport Proteins; Multigene Family; Muscle Proteins; Muscles; Organ Specificity; Phlorhizin; Rats; Subcellular Fractions

Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C.. Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy.. F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA.. These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.

Topics: 2,3-Diketogulonic Acid; Adult; Aged; Animals; Ascorbic Acid; Biological Transport; Buthionine Sulfoximine; Cataract; Cell Culture Techniques; Cytochalasin B; Dehydroascorbic Acid; Enzyme Inhibitors; Epithelial Cells; Fluorine Radioisotopes; Galactose; Glucose; Glutathione; Humans; Hyperglycemia; Lens, Crystalline; Magnetic Resonance Spectroscopy; Male; Middle Aged; Organ Culture Techniques; Oxidative Stress; Rabbits; Rats; Rats, Sprague-Dawley

We investigated the effect of chronic hyperglycemia on glucose transporters in erythrocytes of subjects with and without diabetes mellitus. We found a 22% increase in D-glucose-displaceable cytochalasin-B binding in erythrocyte membranes of diabetic subjects over those of controls (311 +/- 13 vs. 254 +/- 8 pmol/mg protein; P less than 0.001). This increased binding was due to a higher density of binding sites without a significant change in binding affinity. Cytochalasin-B binding to erythrocyte membrane correlated positively with both erythrocyte glycohemoglobin and serum glucose levels, but not with plasma C-peptide levels. The data are compatible with up-regulation of glucose transporters in the erythrocytes of subjects with chronic hyperglycemia. We suspect that this is brought about by increased synthesis and membrane incorporation of the glucose transporter during erythropoiesis.

Topics: Adult; Aged; Blood Glucose; C-Peptide; Chronic Disease; Cytochalasin B; Diabetes Mellitus; Erythrocyte Membrane; Female; Glycated Hemoglobin; Humans; Hyperglycemia; Male; Middle Aged; Monosaccharide Transport Proteins; Reference Values

D-Glucose transport and D-glucose inhibitable [3H]cytochalasin B binding to jejunal basolateral membrane vesicles were measured to investigate the possible association between changes in transport activity seen in hyperglycemia and density of transporter sites. Comparison was made between hyperglycemic animals, noninfused rats, and a group infused with sorbitol. Vascular infusion of D-glucose produced a rapid increase in D-glucose transport followed by a delayed and smaller increase in [3H]cytochalasin B binding. The Vmax for glucose uptake was increased after only 30 min of glucose infusion and continued to rise up to 6 h. Comparison with noninfused and sorbitol-infused controls showed that 2 h of glucose infusion produced a 3.5-fold increase in the Vmax for D-glucose uptake while D-glucose-inhibitable binding of [3H]cytochalasin B was unaffected. Six hours of hyperglycemia resulted in the further stimulation of glucose transport (4.1-fold) and a significant 1.8-fold increase in cytochalasin B binding over that for noninfused animals. Vesicles prepared from animals 4 h after an in vivo injection of cycloheximide showed an 80% reduction in glucose transport with no significant change in the cytochalasin B binding density. These results suggest that D-glucose transport in the basolateral membrane is regulated by a combination of a modulation of carriers already in the membrane and subsequent changes in carrier site density.

Topics: Animals; Binding Sites; Biological Transport; Cycloheximide; Cytochalasin B; Glucose; Hyperglycemia; Insulin; Jejunum; Male; Monosaccharide Transport Proteins; Rats; Rats, Inbred Strains

Topics: Acetates; Adipose Tissue; Animals; Carbon Radioisotopes; Colchicine; Cytochalasin B; Glucose; Hyperglycemia; Male; Mice; Obesity; Tritium; Water