A possible role for the pentose phosphate pathway of spermatozoa in gamete fusion in the mouse.
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Glucose metabolism is essential for successful gamete fusion in the mouse. Although the metabolic activity of the oocyte does not appear to play a significant role in the fusion step, the metabolic role of the spermatozoon is not known. The aim of this study was therefore to characterize the role of glucose metabolism in mouse spermatozoa. Initially, the high-affinity glucose transporter GLUT3 was identified in mouse sperm. In characterizing the glucose metabolism of mouse sperm, we have shown 1) that mouse epididymal spermatozoa have a functional pentose phosphate pathway (PPP), implying that they produce NADPH, which is required for reducing reactions, and ribose 5-phosphate, which is required for nucleic acid synthesis; and 2) that sperm are able to fuse with the oocyte when NADPH is substituted for glucose, suggesting that sperm need to produce NADPH via the PPP in order to be able to achieve fertilization. The existence of an NADPH-regulated event that influences the ability of the sperm to fuse with the oocyte is envisaged. (+info)
Time-dependent and tissue-specific effects of circulating glucose on fetal ovine glucose transporters.
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To determine the cellular adaptations to fetal hyperglycemia and hypoglycemia, we examined the time-dependent effects on basal (GLUT-1 and GLUT-3) and insulin-responsive (GLUT-4) glucose transporter proteins by quantitative Western blot analysis in fetal ovine insulin-insensitive (brain and liver) and insulin-sensitive (myocardium, skeletal muscle, and adipose) tissues. Maternal glucose infusions causing fetal hyperglycemia resulted in a transient 30% increase in brain GLUT-1 but not GLUT-3 levels and a decline in liver and adipose GLUT-1 and myocardial and skeletal muscle GLUT-1 and GLUT-4 levels compared with gestational age-matched controls. Maternal insulin infusions leading to fetal hypoglycemia caused a decline in brain GLUT-3, an increase in brain GLUT-1, and a subsequent decline in liver GLUT-1, with no significant change in insulin-sensitive myocardium, skeletal muscle, and adipose tissue GLUT-1 or GLUT-4 concentrations, compared with gestational age-matched sham controls. We conclude that fetal glucose transporters are subject to a time-dependent and tissue- and isoform-specific differential regulation in response to altered circulating glucose and/or insulin concentrations. These cellular adaptations in GLUT-1 (and GLUT-3) are geared toward protecting the conceptus from perturbations in substrate availability, and the adaptations in GLUT-4 are geared toward development of fetal insulin resistance. (+info)
Developmental regulation of genes mediating murine brain glucose uptake.
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We examined the molecular mechanisms that mediate the developmental increase in murine whole brain 2-deoxyglucose uptake. Northern and Western blot analyses revealed an age-dependent increase in brain GLUT-1 (endothelial cell and glial) and GLUT-3 (neuronal) membrane-spanning facilitative glucose transporter mRNA and protein concentrations. Nuclear run-on experiments revealed that these developmental changes in GLUT-1 and -3 were regulated posttranscriptionally. In contrast, the mRNA and protein levels of the mitochondrially bound glucose phosphorylating hexokinase I enzyme were unaltered. However, hexokinase I enzyme activity increased in an age-dependent manner suggestive of a posttranslational modification that is necessary for enzymatic activation. Together, the postnatal increase in GLUT-1 and -3 concentrations and hexokinase I enzymatic activity led to a parallel increase in murine brain 2-deoxyglucose uptake. Whereas the molecular mechanisms regulating the increase in the three different gene products may vary, the age-dependent increase of all three constituents appears essential for meeting the increasing demand of the maturing brain to fuel the processes of cellular growth, differentiation, and neurotransmission. (+info)
An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes.
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The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane. (+info)
Glucose transporter Glut3 is targeted to secretory vesicles in neurons and PC12 cells.
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In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells. (+info)
Regulation of GLUT-3 glucose transporter in the hippocampus of diabetic rats subjected to stress.
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Previous studies from our laboratory have demonstrated that chronic stress produces molecular, morphological, and ultrastructural changes in the rat hippocampus that are accompanied by cognitive deficits. Glucocorticoid attenuation of glucose utilization is proposed to be one of the causative factors involved in stress-induced changes in the hippocampus, producing an energy-compromised environment that may make hippocampal neuronal populations more vulnerable to neurotoxic insults. Similarly, diabetes potentiates neuronal damage in acute neurotoxic events, such as ischemia and stroke. Accordingly, the current study examined the regulation of the neuron-specific glucose transporter, GLUT-3, in the hippocampus of streptozotocin-induced diabetic rats subjected to restraint stress. Diabetes leads to significant increases in GLUT-3 mRNA and protein expression in the hippocampus, increases that are not affected by stress. Collectively, these results suggest that streptozotocin-induced increases in GLUT-3 mRNA and protein expression in the hippocampus may represent a compensatory mechanism to increase glucose utilization during diabetes and also suggest that modulation of GLUT-3 expression is not responsible for glucocorticoid impairment of glucose utilization. (+info)
Enhanced expression of glucose transporter GLUT3 in tumorigenic HeLa cell hybrids associated with tumor suppressor dysfunction.
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Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced Km by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUT3 together with GLUT1 of 70 kDa was also evident in three gamma-ray-induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2-deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N-glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2-deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed. (+info)
Specificity of ascorbate analogs for ascorbate transport. Synthesis and detection of [(125)I]6-deoxy-6-iodo-L-ascorbic acid and characterization of its ascorbate-specific transport properties.
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Cellular ascorbic acid accumulation occurs in vitro by two distinct mechanisms: transport of ascorbate itself or transport and subsequent intracellular reduction of its oxidized product, dehydroascorbic acid. It is unclear which mechanism predominates in vivo. An easily detectable compound resembling ascorbate but not dehydroascorbic acid could be a powerful tool to distinguish the two transport activities. To identify compounds, 21 ascorbate analogs were tested for inhibition of ascorbate or dehydroascorbic acid transport in human fibroblasts. The most effective analogs, competitive inhibitors of ascorbate transport with K(i) values of 3 microM, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate. No analog inhibited dehydroascorbic acid transport. Using substitution chemistry, [(125)I]6-deoxy-6-iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection methods were developed for radiolabeled and nonradiolabeled compounds, and transport kinetics of both compounds were characterized. Transport was sodium-dependent, inhibited by excess ascorbate, and similar to that of ascorbate. Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate was investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3. Oxidation of ascorbate or its analog in media increased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorbate, although an effective inhibitor of ascorbate transport, either in its reduced or oxidized form was not a substrate for dehydroascorbic acid transport. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate provide a new means for discriminating dehydroascorbic acid and ascorbate transport in ascorbate recycling. (+info)