Kinetic characteristics of chloroplast glucose transport

Influx of labelled D-glucose into isolated spinach (Spinacia oleracea L. cv. Melody hybrid) chloroplasts was initially rapid followed by a period of slower influx. The stroma glucose concentration attained equilibrium rapidly with low external glucose concentrations and the two were linearly proportional. The period of slower influx resulted from conversion of glucose to acidic products that remained trapped in the chloroplast. As the external glucose concentration increased, the stroma glucose concentration increased less and less, attaining a maximal concentration of 72 mol m(-3). The maintenance of an equilibrium stroma glucose concentration lower than that in the external medium is evidence that plastid glucose efflux involves secondary active transport. The equilibrium stroma glucose concentration increased in response to light and protonophoric uncouplers. It is proposed that glucose efflux is coupled with a proton and the stroma glucose concentration equilibrates in response to the proton gradient across the membrane. To determine if glucose is a significant product of starch mobilization, chloroplasts were isolated from spinach leaves labelled with 14CO2 during the preceding light period. Chloroplasts degraded starch at the same rate as the intact leaf. Glucose, maltose, and isomaltose were the principal labelled products that appeared in the medium during starch mobilization. The glucose concentration in the chloroplast was 2 mol m(-3), which is similar to the measured Km for zero trans efflux. The data support the role of the glucose translocator as an important component in the pathway for sucrose synthesis at night.     

Four grams of glucose

Four grams of glucose circulates in the blood of a person weighing 70 kg. This glucose is critical for normal function in many cell types. In accordance with the importance of these 4 g of glucose, a sophisticated control system is in place to maintain blood glucose constant. Our focus has been on the mechanisms by which the flux of glucose from liver to blood and from blood to skeletal muscle is regulated. The body has a remarkable capacity to satisfy the nutritional need for glucose, while still maintaining blood glucose homeostasis. The essential role of glucagon and insulin and the importance of distributed control of glucose fluxes are highlighted in this review. With regard to the latter, studies are presented that show how regulation of muscle glucose uptake is regulated by glucose delivery to muscle, glucose transport into muscle, and glucose phosphorylation within muscle.     

Catabolite inactivation of the maltose transporter in nitrogen-starved yeast could be due to the stimulation of general protein turnover

Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter. The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available. Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole. We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose. This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose. It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.     

Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent K_m for glucose of approximately 1.1 - 2.9 mM, consistent with the function of Gif as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z mobilis glucokinase gene, in addition to glf, did not affect the K_m of Glf for glucose in recombinant E. coli The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Gif for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.     

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations.     

Digitoxose and the existence of a glucoreceptor in the beta cells of the islets of langerhans of the rat

On the basis that digitoxin is the only drug reported up to now that stimulating insulin release per se, partially inhibits glucose stimulated insulin release, in this work the idea that the competition glucose-digitoxin may be at the level of a hypothetical glucoreceptor in the beta cells (binding of the glycoside sugar molecule) has been investigated. Our findings indicate that the integrity of the chemical structure of digitoxin is needed to exert its pharmacological action on insulin release. We also demonstrate that digitoxose inhibits glucose stimulated insulin release without inhibiting the glucose oxidation rates of the islets. The discovery of a sugar (digitoxose) that inhibitis the insulin secretory response to glucose without inhibiting glucose metabolism is a strong evidence that the action of glucose itself is blocked at the level of glucoreceptors located in the cell membrane, which appear to be the regulator sites of insulin release.     

Effects of hypo and hyperthyroidism on the response to glucose loading of blood glucose, ketones and insulin and liver glycogen as studied in the fasted rat

After receiving an i.p. glucose load, 24 h fasted thyroidectomized rats showed a progressive increase in blood glucose and a slow decrease in blood ketone bodies. Both liver glycogen and plasma insulin levels showed no differences within 60 min of the glucose administration. It is suggested that the glucose intolerance in these animals is partly due to an insulin deficiency. Thyroidectomized rats treated daily with 25 microgram of L-thyroxine/100 g body weight for 40 days responded to the glucose test with a supranormal and more persistent elevation of blood glucose but with a faster and a greater fall in blood ketone bodies, as compared to controls. Sixty min after the glucose loading, liver glucogen levels were lower and plasma insulin were slightly higher than controls. It is suggested that a diminished extraction of glucose during transhepatic passage can be responsible for the impaired glucose tolerance observed in the hyperthyroid animals.     

Regulation of hepatic glucose metabolism by translocation of glucokinase between the nucleus and the cytoplasm in hepatocytes.

We studied the role of glucokinase translocation between the nucleus and the cytoplasm in hepatocytes. In cultured hepatocytes, both the translocation of glucokinase from the nucleus to the cytoplasm and the rate of glucose phosphorylation were increased when cells were incubated with high concentrations of glucose. The addition of low concentrations of fructose, which is known to stimulate glucose phosphorylation, stimulated both glucokinase translocation and glucose phosphorylation. There was a good correlation between the increase in cytoplasmic glucokinase induced by fructose and that in the glucose phosphorylation rate induced by fructose. Furthermore, we observed a linear relationship between cytoplasmic glucokinase activity and rate of glucose phosphorylation over various glucose concentrations in the absence or presence of fructose. These results indicate that glucose phosphorylation in hepatocytes depended on glucokinase in the cytoplasmic compartment--that is, the increase in the rate of glucose phosphorylation was due to the increase in translocation of glucokinase out of the nucleus. Also, oral administration of glucose, fructose, or glucose plus fructose to 24-h fasted rats induced translocation of glucokinase in the liver. All of these results indicate that hepatic glucose metabolism is regulated by the translocation of glucokinase.     

Control of glucose metabolism in the perinatal period

The central importance of glucose as a fuel for energy metabolism and growth of the fetus is clear as is the role of insulin in coordinating its utilisation by many fetal tissues. What is less clear is the qunatitative nature of the interaction between the fetus and placenta in organising glucose metabolism. Increasingly there is evidence that the fetus coordinates some of the supply of glucose to the placenta and that this is particularly important when uterine blood flow is reduced. It is unclear how this is regulated, but substrate cycles of glucose and lactate appear to make a significant contribution to carbohydrate metabolism in fetus and placenta. Another area as yet unresolved in the control of fetal glucose metabolism is the coordination of the changes that occur around the time of birth. Notable of these is the activation of glycogen mobilisation and of glucose synthesis and changes in the setting of glucose regulatory mechanism. These are briefly reviewed.     

Transport of D-allose by isolated fat-cells: An effect of adenosine triphosphate on insulin stimulated transport

D-allose, a glucose analogue, is not metabolized by isolated fatcells and its distribution space at equilibrium in the cells is the same as that of tritiated water. Uptake of allose is inhibited by glucose and 3-0-methylglucose, stimulated by insulin and virtually eliminated by cytochalasin B. Counter transport of allose out of fat-cells against a concentration gradient can be induced by exogenous glucose but not by pyruvate. It is concluded that allose is transported into fat-cells by the same carrier mediated transport system as glucose and that it is a suitable analogue with which to study the glucose transport system. Insulin stimulated allose transport, into or out of the cell, but not basal transport, is inhibited by a brief exposure of isolated fat-cells to exogenous ATP or ADP (but not AMP or AMP-PNP). The antilipolytic effect of insulin is not affected. The ATP inhibition is slowly reversible. It is suggested that ATP phosphorylates a membrane component and thereby blocks transmission of signal from the insulin receptor to the carrier system. Indirect evidence suggests that ATP does not alter the affinity of the insulin or glucose binding sites. Insulin decreases the K_m of glucose metabolism to CO₂ and lipid in isolated fat-cells and increases the V_max. However, the hormone has no effect on the K_i of glucose as an inhibitor of allose transport. The glucose analogue, 3-0-methylglucose, also inhibits both glucose metabolism and allose transport. The K_i for both these processes is similar and is not affected by insulin. These results support the view that the effect of insulin on glucose transport is to raise the V_max without a change in the K_m. It appears further that sugar transport is not the major rate limiting step in metabolism at high glucose concentrations in the absence of insulin, or at most glucose concentrations in the presence of the hormone.     

Absorption of an oral glucose load in the dog

Absorption of glucose from the gut was estimated in trained unanesthetized dogs given a glucose load of 1-25 g (14C)glucose by stomach tube. The rate of absorption of glucose was calculated from the concentration and specific activity of glucose in the portal vein and in an "arterialized" peripheral vein. When the rate was integrated over time it was found that 94 +/- 4% of the administered glucose was recovered from the portal vein as glucose; this was unrelated to the size of the glucose load. It is concluded that absorption does not entail a significant loss or conversion to glucose metabolites.     

Effect of glucose feeding on the glycosylation quality of antibody produced by a human cell line,F2N78, in fed-batch culture

The human cell line rF2N78 produces an antibody with a high galactosylation ratio which resembles human IgG. However, it has been observed that the aglycosylated antibody starts to appear when glucose is depleted. To determine whether glucose depletion is a main cause for aglycosylation of the antibody, fed-batch cultures of rF2N78 cells were performed using different feeding cocktails (glucose only, nutrient feeding cocktail without glucose, and nutrient feeding cocktail with glucose). In the fed-batch culture with nutrient feeding cocktail without glucose, aglycosylated antibody was produced in a later phase of culture, when glucose was depleted. Approximately 44 % of antibodies produced were aglycosylated at the end of culture. In contrast, aglycosylated antibody was not produced in cultures with glucose feeding. The expression levels of oligosaccharyl transferases determined by Western blot analysis were similar among the cultures, suggesting that aglycosylation of the antibody was not due to altered expression of oligosaccharyl transferases under glucose-deficient conditions. Thus, it is likely that glucose deficiency led to insufficiency of the precursor for glycosylation and induced aglycosylation of the antibody. Taken together, glucose feeding in fed-batch cultures successfully prevented occurrence of aglycosylated antibody during the cultures, confirming that glucose depletion is a main cause for aglycosylation of antibody.     

Bifurcate effects of glucose on caspase-independent cell death during hypoxia

We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.     

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake.     

Glucose diffusion in pancreatic islets of Langerhans.

We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet beta-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10 mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1-2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose.