Which of the following describes why a glucose transporter is needed to move glucose into the cell?

Introduction

Glucose transport supplies fuel that is needed for energy metabolism by most mammalian cells. Glucose is a very common metabolic substrate that is used both as a fuel and a signaling molecule. The supply of glucose is especially important for certain cells, such as brain neurons, which have a high metabolic rate supported by an obligate consumption of glucose as fuel in most circumstances. Transport of glucose is regulated by a variety of factors including those associated with several aspects of cellular stress. Transport proteins that accomplish glucose transport are modulated in their expression, cellular distribution, synthesis, and half-lives by stress-related factors. Such factors include stress hormones, a variety of metabolic stresses, such as cellular energy demand, metabolic poisons, inflammation, and stress-related kinase signaling, endoplasmic reticulum (ER) stress, and chronic diseases. The net effect of such regulation may be favorable and serve to ensure appropriate distribution of glucose fuel to tissues during stress that most require this particular fuel. However, some adaptations of glucose transport promote or worsen diseases such as cancers.

Effect on Glucose Transporters

Glucose transport by astrocytes is mainly brought about through the energy-independent facilitative glucose transporter GLUT-1. The inhibition of gap junctional communication promotes a rapid translocation of GLUT-1 from an intracellular pool to the plasma membrane, which may be responsible for the increase in the Vmax for glucose uptake found when gap junctional communication is inhibited. Similar changes in GLUT-1 distribution have been reported in response to some growth factors, indicating that this system may be designed to rapidly increase glucose uptake to meet cellular metabolic requirements.

In addition to this short-term regulation, the level of GLUT-1 is increased after 24 h of treatment with inhibitors of gap junctional communication, indicating that the inhibition of gap junctional communication also triggers a long-term regulation of glucose transport. In fact, GLUT-3, which is not normally present in astrocytes, although its expression can be induced under several circumstances, is induced after the inhibition of gap junctional communication. The upregulation of GLUT-3 has been associated with an increase in glucose uptake because this isoform exhibits a higher capacity for glucose transport than does GLUT-1. In fact, the Km for glucose uptake is significantly reduced after treatment with the inhibitors of gap junctional communication.

Glucose

Transplacental glucose transport is mediated by facilitated diffusion through specific GLUTs. A higher density of GLUTs in the MVM, together with the greater surface area, allows rapid glucose uptake into the syncytiotrophoblast and thus provides a maximal gradient for transfer to the fetus across the BM.189 Several GLUT isoforms are expressed in the human syncytiotrophoblast, and GLUT1 is the main isoform mediating glucose transport across the placenta throughout pregnancy.189 The placental expression of GLUT1 protein and glucose transport activity are unaffected by IUGR; therefore, fetal hypoglycemia in IUGR is unlikely due to changes in placental GLUT expression or activity.189,190

Glucose uptake and glycogen

Glucose transport in myocytes is driven by the translocation of monosaccharide transporters (GLUT-4 and GLUT-1) to the sarcolemma. Insulin-mediated GLUT-4 is the major glucose transporter in cardiac and skeletal muscle [7]. In contrast, GLUT-1 shows a much lower glucose uptake rate and rather mediates general glucose uptake in most tissues. Additionally, intracellular glycogen stores are another potential source of glucose. Cardiac glycogen stores are small compared to other tissues such as liver or skeletal muscle. There is a rapid turnover of glucose to glycogen for storage and of glycogen to glucose as substrate in glycolysis (see Chapter 5). Cardiomyocytes therefore present with quite stable glycogen concentration. However, high extracellular glucose concentrations increase the glycogen pool [8]. In contrast, elevated amounts of AMP, inorganic phosphate, and a fall in ATP activate glycogenolysis and result in enhanced substrate supply for glycolysis [9].

Overview of Glucose Transport Regulation

Glucose transport is a highly regulated process that varies considerably from one cell type to another. Some cells, such as red blood cells and brain neurons, have obligate consumption of glucose. For other cells, a facultative use of glucose exists, permitting other metabolic fuels, such as fatty acids, to supply the bulk of local energy requirements. Much evidence suggests that the regulation of glucose transport is also isoform specific. Regulation of transport occurs in response to altered energy requirements of tissues, so it is not surprising that one form of cellular stress, energy lack, is a potent regulator of glucose transport and transporter expression by different tissues. Regulation of GLUT proteins may occur by variation of the amounts of synthesis or degradation of the GLUT protein or mRNA. An increased transcription of GLUT mRNA or other regulatory effects on GLUTs may occur as a result of stress hormones, such as glucocorticoids and epinephrine. In some tissues, these hormones have differing effects on expression of GLUTs or their transport activity, emphasizing their tissue-specific regulation. The effects of growth factors, physiological factors often involved in stress responses, increase GLUT1 transcription.

3.1 Effect of hypoxia on glucose transporters

Glucose transport in alveolar epithelial cells takes place through different pathways. Based on in vivo and in vitro studies, glucose enters alveolar epithelial cells mostly by a Na+-independent carrier-mediated process (Clerici et al., 1991). The ubiquitous glucose transporter GLUT1, which is responsible for basal glucose uptake in most tissues, is predominant in ATII cells (Saumon and Makhloufi, 1997). However, the presence of an apical Na+-dependent D-glucose transporter in alveolar epithelial cells was reported in in vivo studies but the results are less consistent in vitro (O’Brodovich et al., 1991; Clerici et al., 1991).

In alveolar epithelial cells, hypoxia increases anaerobic glycolysis by upregulating both glucose transport at the membrane level (Ouiddir et al., 1999) and glycolytic enzymes. In cultured ATII cells, hypoxia induced an increase in glucose influx, measured by the uptake of deoxy-D-glucose (DG), a glucose analog that is transported by the facilitative glucose transporter. This stimulation of glucose influx was associated with an increase in mRNA and protein levels of GLUT 1. Several lines of evidence suggest that the hypoxia-induced increase of DG uptake was related to upregulation of GLUT 1: (i) the time course for the hypoxia-induced increase in DG influx, with no substantial change before 6 h of hypoxia; (ii) the prevention of this effect by cycloheximide, an inhibitor of translation; and (iii) the parallel recovery during reoxygenation of DG uptake and the level of GLUT 1 mRNA. Comparison of ATII cells with other hypoxic tolerant cells showed that they resemble lung endothelial cells (Loike et al., 1992), but differ from heart and skeletal muscle in which upregulation of glucose transport occurs in less than one hour with no change of GLUT 1 mRNA level (Cartee et al., 1991). In ATII cells, Na-dependent glucose transport is weakly expressed in normoxia, and Ouiddir and colleagues showed that it was not stimulated by hypoxia (Ouiddir et al., 1999) (Fig. 2.2).

The O2 sensing mechanisms whereby hypoxia regulates the level GLUT1 mRNA in ATII cells are not univocal and result from both a reduced O2, concentration per se and inhibition of oxidative phosphorylation (Ouiddir et al., 1999). This distinction has been made through studies with specific chemical agents that mimic the actions of the different components of the hypoxic response (Behrooz and Ismail, 1997). Most hypoxia-regulated genes involve a ferroprotein sensor and an important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals. For instance, in the presence of O2, cobalt chloride simulates the effect of lowered O2 concentration since it substitutes for O2 in the heme protein. In normoxic ATII cells, GLUT1 mRNA levels and glucose uptake are increased by cobalt chloride, strongly supporting the hypothesis that upregulation of GLUT1 gene is dependent on O2 deprivation itself. However, inhibition of oxidative phosphorylation by sodium azide in normoxic ATII cells is as effective as hypoxia for stimulating glucose transport and increasing GLUT1 mRNA levels. That hypoxia-induced GLUT1 gene upregulation results from two different mechanisms was also previously reported in a cell clone derived from a hepatoma (Behrooz and Ismail, 1997). However, the temporal and spatial contribution of these two mechanisms in hypoxia-induced upregulation of GLUT1 mRNA level in ATII cells remains to be determined.

8.11.2.3 Passive Glucose Transport

Glucose transport kinetics illustrates the salient features encountered in facilitated transport. Net glucose transport rates, in human erythrocytes, are normally measured from a (cis) solution containing glucose into the nominally glucose-free (trans) solution on the other side of the cell membrane. The cis glucose concentration at which the net transport rate is half maximal is the Km, termed the Km for zero trans inflow or import. At 24 °C, the erythrocyte glucose transporter, GLUT1, has approximately a 10-fold lower affinity for d-glucose, Km≈10–15 mM at the inside trans face for net export than on the cis outside (Km=1–2 mM) for net import of glucose (zero-trans net flux). At 4 °C, the affinities are higher and the asymmetry is larger than at 24 °C15 (Figure 3(a)). Detailed Balance is conserved in the cyclic carrier model by assigning the product of the clockwise rates equal to the product of all anticlockwise rates for the four-node cycle (Figures 1 and 4(b))

[2]k12k23k34k41 =k21k14k43k32

It may be deduced4b from the Detailed Balance requirement that the ratio Vm/Km for the asymmetric operational transport:

[3]Vmimport/K mimport=Vmexport/Kmexport

Vm is the maximal rate of glucose transport into or out of the cell. While this simple Haldane relationship is a necessary corollary of the cyclic carrier model, it has often been observed that the experimentally derived import and export parameters, Vm and Km, do not conform to the Haldane equality. For this reason, alone, the cyclic carrier model is an inadequate description of glucose transport,15s,16 and alternative models to describe facilitated glucose transport have been sought.

4.1 GLUT1/GLUT4 and HK2

Glucose uptake mediated by GLUTs is the first step of glucose metabolism. p53 limits this first step by directly binding to the GLUT1 and GLUT4 promoters, consequently attenuating their gene expression levels (Schwartzenberg-Bar-Yoseph et al., 2004). p53 that has a tumor-associated mutation in the DNA-binding domain fails to suppress the promoter activity of GLUT1 and GLUT4 (Schwartzenberg-Bar-Yoseph et al., 2004), thereby increasing cellular glucose levels. Mutant p53, but not wild-type p53, also promotes GLUT1 translocation to the plasma membrane and enhances glucose uptake (Zhang et al., 2013).

p53 also downregulates the gene expression of hexokinase-2 (HK2), an enzyme that converts glucose to G6P. The loss of the p53 gene results in an increase in HK2 mRNA levels by transcript stabilization (Wang et al., 2014). Moreover, tumor-associated mutant p53 enhances HK2 gene expression through two p53 response elements of the HK2 promoter region (Mathupala et al., 1997).

Glucose Metabolism and Foetal Growth

Glucose transport across the placenta occurs through facilitated diffusion, aided by specific glucose transporters. The GLUT1 glucose transporter, present on both microvilli and basal membranes of the syncytial barrier, is the primary isoform involved in the transplacental movement of glucose, and is the rate-limiting step. It has been noted that, in diabetic pregnancies, there is an increase in basal GLUT1 expression and activity, which is present despite near-normal glycaemia at term, with significant consequences for the maternal–foetal flux of glucose [74,75].

Choice of Functional Probes for the Study of Glucose Transporters

Glucose transport in cell populations is often studied with isotope-labeled hexoses, both in culture and in vivo. After entering cells, labeled glucose is rapidly metabolized to lactate and CO2. Because both products can leave the cell very rapidly, glucose uptake is not a reliable measure of glucose transport. 3-O-Methyl-d-glucose (3MG) is a good nonmetabolized substrate for GLUTs, but in brain cells it is transported so quickly that accurate measurement of initial rates at room temperature or higher is not easy. 2-Deoxyglucose (DG) is transported with high efficiency and then phosphorylated by hexokinase but does not proceed further. Although DG is by far the most popular substrate used to characterize GLUTs in the brain and elsewhere, DG data must be interpreted with caution, for depending on the times chosen for assay and the relative number of GLUTs and hexokinase, DG uptake rates may reflect the phosphorylation step rather than the transport step. Contrary to common wisdom, a linear time course of uptake should not be considered proof of the contrary. The transport of sugars into single cells can also be studied by fluorescence microscopy. A fluorescent derivative of DG, 6-NBDG, is a hexose that binds GLUTs with apparent affinity similar to that of glucose but translocates with very low probability, a useful property which allows real-time monitoring of uptake rates. 2-NBDG behaves similarly but it is phosphorylated by hexokinase. As with DG, whether changes in 2-NBDG uptake reflect modulation of transport or phosphorylation must be determined under each experimental condition. Finally, GLUTs can be studied by following osmotically obliged water movements in calcein-loaded cells using confocal microscopy. This technique works best for cells endowed with a high-capacity, low-affinity transporter and provides direct estimates of Vmax values.