The effect of phenazine methosulphate on intermediary pathways of glucose metabolism in the lens at different glycaemic levels

In this study changes in alternative pathways of glucose metabolism are examined in the rat lens using radiolabelled glucose in a 1 hr in vitro incubation of 50 mM or 10 mM glucose with or without 0.1 mM phenazine methosulphate (PMS). PMS which reoxidizes NADPH ensures that the pentose phosphate pathway (PPP) is not limited by the supply of NADP+. The data shows that maximal activation of the PPP (with PMS) is 40% greater at high glucose concentrations than normal glucose. This difference in maximal stimulation may be explained by the increase glucose uptake in the hyperglycaemic incubation. In the high-glucose incubation with PMS, hexokinase activity and the glucose 6-phosphate pool is not limiting for the PPP. Under these conditions, PMS alter the NAD+/NADH and NADP+/NADPH ratio. The change in the redox state alters the flux through the polyol pathway, the glycerol 3-phosphate shuttle and the glycolytic control sites, glyceraldehyde 3-phosphate, pyruvate and lactate dehydrogenases. These results are discussed in relation to hyperglycaemia-induced oxidative stress.     

Selective outgrowth and differential tropism of amacrine and photoreceptor axons to cell targets during early development in vitro

Plastids of nongreen tissues import carbon as a source of biosynthetic pathways and energy. Within plastids, carbon can be used in the biosynthesis of starch or as a substrate for the oxidative pentose phosphate pathway, for example. We have used maize endosperm to purify a plastidic glucose 6-phosphate/phosphate translocator (GPT). The corresponding cDNA was isolated from maize endosperm as well as from tissues of pea roots and potato tubers. Analysis of the primary sequences of the cDNAs revealed that the GPT proteins have a high degree of identity with each other but share only approximately 38% identical amino acids with members of both the triose phosphate/phosphate translocator (TPT) and the phosphoenolpyruvate/phosphate translocator (PPT) families. Thus, the GPTs represent a third group of plastidic phosphate antiporters. All three classes of phosphate translocator genes show differential patterns of expression. Whereas the TPT gene is predominantly present in tissues that perform photosynthetic carbon metabolism and the PPT gene appears to be ubiquitously expressed, the expression of the GPT gene is mainly restricted to heterotrophic tissues. Expression of the coding region of the GPT in transformed yeast cells and subsequent transport experiments with the purified protein demonstrated that the GPT protein mediates a 1:1 exchange of glucose 6-phosphate mainly with inorganic phosphate and triose phosphates. Glucose 6-phosphate imported via the GPT can thus be used either for starch biosynthesis, during which process inorganic phosphate is released, or as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates.     

Alteration of glucose uptake in cultured human corneal endothelial cells grown in high glucose media via cAMP-dependent pathway

In this study, cultured human corneal endothelial cells were incubated in media containing various concentrations of glucose at 5 mM, 10 mM, and 25 mM for 2 days. Then, the cellular 2-deoxyglucose uptake and cAMP concentration of cultured human corneal endothelial cells were measured. The results indicated that the activity of cellular glucose uptake of nmole/min/mg protein was decreased gradually from 0.18 (5 mM), 0.10 (10 mM), 0.07 (20 mM) to 0.06 (25 mM) after 2 days incubation with a high concentration of glucose. The glucose uptake in insulin-treated human corneal endothelial cells also exhibited a similar declining effect in high glucose media from 0.30 (5 mM), 0.11 (10 mM), 0.08 (20 mM) to 0.05 (25 mM). The cAMP concentration in human corneal endothelial cells was measured in the presence of high glucose media. It was indicated that the cAMP concentrations of pmole/well in both insulin-treated and non-insulin treated cells were also decreased after increasing the glucose concentration in the media from 73 (5 mM) to 20 (25 mM) and 101 (5 mM) respectively. The cAMP concentration in insulin-treated cells was less than in non-insulin treated cells. This decreasing effect was significantly reversed by the addition of 1 mM dibutyryl-cAMP to the cells for 1 hour in both groups. These results suggest that the diabetic state may decrease the 2-deoxyglucose uptake in human corneal endothelial cells via cAMP-dependent pathway.     

The three-dimensional structure of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides refined at 2.0 A resolution

BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme of the pentose phosphate pathway. Normally the pathway is synthetic and NADP-dependent, but the Gram-positive bacterium Leuconostoc mesenteroides, which does not have a complete glycolytic pathway, also uses the oxidative enzymes of the pentose phosphate pathway for catabolic reactions, and selects either NAD or NADP depending on the demands for catabolic or anabolic metabolism. RESULTS: The structure of G6PD has been determined and refined to 2.0 A resolution. The enzyme is a dimer, each subunit consisting of two domains. The smaller domain is a classic dinucleotide-binding fold, while the larger one is a new beta+ alpha fold, not previously seen, with a predominantly antiparallel nine-stranded beta-sheet. There are significant structural differences in the coenzyme-binding domains of the two subunits, caused by Pro 149 which is cis in one subunit and trans in the other. CONCLUSIONS: The structure has allowed us to propose the location of the active site and the coenzyme-binding site, and suggests the role of many of the residues conserved between species. We propose that the conserved Arg46 would interact with both the adenine ring and the 2'-phosphate of NADP. Gln47, which is not conserved, may contribute to the change from NADP to dual coenzyme specificity. His178, in a nine-residue peptide conserved for all known sequences, binds a phosphate in the active site pocket. His240 is the most likely candidate for the base to oxidize the 1-hydroxyl group of the glucose 6-phosphate substrate.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp, encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO2. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp.     

Changes in glucose transporter 2 and carbohydrate-metabolizing enzymes in the liver during cold preservation and warm ischemia

In order to examine glucose metabolism in liver grafts during cold preservation (24 and 48 hr), warm ischemia (60 and 120 min), a combination of the two and reperfusion, the amount of protein and mRNA of glucose transporter 2 and the activities of enzymes in glycolysis (glucokinase, phosphofructokinase, pyruvatekinase), gluconeogenesis (glucose 6-phosphatase, fructose 1,6-bisphosphatase), and the pentose phosphate pathway (glucose 6-phosphate dehydrogenase) were measured. It appeared that glucose transport, the pentose phosphate pathway, and gluconeogenesis were maintained during cold preservation and warm ischemia. The activity of glucokinase significantly decreased from the control value of 1.33 +/- 0.23 IU/g protein to 0.70 +/- 0.17 (24 hr, P<0.05) and 0.57 +/- 0.12 (48 hr, P<0.01) only during cold preservation. However, the activity of phosphofructokinase significantly decreased from the control value of 4.37 +/- 0.06 IU/g protein to 2.67 +/- 0.15 (60 min, P<0.0001) and 1.53 +/- 0.06 (120 min, P<0.0001) only during warm ischemia. This indicates that glycolysis deteriorates during both cold preservation and warm ischemia and demonstrates further that the balance between glycolysis and gluconeogenesis shifts to gluconeogenesis. Even when cold preservation was combined with warm ischemia, the activity of glucokinase decreased only during cold preservation and the activity of phosphofructokinase decreased only during warm ischemia. Furthermore, these changes were time-dependent. It is suggested that they can be used as a clock to measure the durations of cold preservation and warm ischemia separately and that the magnitude of an ischemic injury to a liver and a liver graft's viability can be indirectly estimated before transplantation.     

Glycaemic regulation of glucose transporter expression and activity in the human placenta

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.     

Disappearance of the Vicia villosa-positivity from the perineuronal net containing chondroitin proteoglycan after chondroitinase digestion

The effects of thyroid status on glycolysis using 10, 20, and 40 mM glucose have been examined in hepatocytes derived from hypothyroid, euthyroid, and hyperthyroid rats. For any given concentration of added glucose, total glycolytic rates, as measured by the release of tritium from [6-3H]glucose, were similar in all thyroid states. The aerobic component of glycolysis, where cytoplasmically generated reducing equivalents are transferred to the mitochondria for oxidation, was the major component in the hyperthyroid state, at all concentrations of glucose. In contrast, the aerobic proportion of glycolysis in the hypothyroid and euthyroid states decreased with increasing concentration of added glucose and the anaerobic component became dominant above 20 mM glucose. Cytoplasmic reducing equivalents generated during aerobic glycolysis were transferred to the mitochondria via both the glycerol 1-phosphate and malate/aspartate shuttles in each thyroid state, even though the former shuttle was considerably depressed in the livers of hypothyroid rats. Both asparagine and aminooxyacetate had only minor effects on the rate of glycolysis, but aminooxyacetate depressed the contribution of aerobic glycolysis whereas asparagine had relatively little influence. The respiration rate in the presence of 40 mM glucose was twice as high in hepatocytes from hyperthyroid rats as in cells from hypothyroid animals, and 1.4 times as high as in hepatocytes from euthyroid rats. Smaller stimulations were observed with lower concentrations of added glucose. Furthermore, the increase in respiratory rate over the endogenous value, induced by 10 mM glucose, was six times higher in cells from hyperthyroid rats than in hepatocytes from hypothyroid animals and 2.7 times higher than that observed with cells from euthyroid rats. The insensitivity of glycolysis to thyroid status in contrast to the marked response of respiration provides additional support for the view that the stimulation of metabolism by thyroid hormone is mediated primarily by its action on mitochondrial processes.     

Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)     

Absence of short-term regulation over gluconeogenesis by glucose in the insect Manduca sexta L

In vivo gluconeogenesis from (3-13 C)alanine was evident in terminal instar Manduca sexta larvae from the selective fractional 13C enrichment in trehalose, a disaccharide of glucose and the major blood sugar of insects. De novo glucose synthesis was observed in insects fed a low carbohydrate diet for 1 or more days. Gluconeogenesis was not inhibited by a single injection of glucose nor by short-term feeding on glucose-supplemented diet. Reduced fractional 13C enrichment in trehalose was demonstrated upon glucose administration, but was explained by isotopic dilution following direct synthesis of trehalose from the unlabeled glucose. Isotopic dilution was also quantified by analysis of the 13C labeling pattern in trehalose synthesized following injection of (1,2-13C2)glucose. The results suggest the absence of short-term regulation over gluconeogenesis by glucose and may partially explain why blood sugar level in M. sexta and other insects fluctuates over a wide concentration range. Although glucose had no observable effects on gluconeogenesis, injection of or feeding glucose resulted in a significantly increased activity of the pentose phosphate pathway.     

In vivo and in vitro regulation of hepatic glucagon receptor mRNA concentration by glucose metabolism

We have recently cloned the murine glucagon receptor (GR) gene and shown that it is expressed mainly in liver. In this organ, the glucagon-GR system is involved in the control of glucose metabolism as it initiates a cascade of events leading to release of glucose into the blood stream, which is a main feature in several physiological and pathological conditions. To better define the metabolic regulators of GR expression in liver we analyzed GR mRNA concentration in physiological conditions associating various glucose metabolic pathways in vivo and in vitro in the rat and in the mouse. First, we report that the concentration of the GR mRNA progressively increased from the first day of life to the adult stage. This effect was abolished when newborn rodents were fasted. Second, under conditions where intrahepatic glucose metabolism was active such as during fasting, diabetes, and hyperglycemic clamp, the concentration of GR mRNA increased independent of the origin of the pathway that generated the glucose flux. These effects were blunted when hyperglycemia was corrected by phlorizin treatment of diabetic rats or not sustained during euglycemic clamp. In accordance with these observations, we demonstrated that the glycolytic substrates glucose, mannose, and fructose, as well as the gluconeognic substrates glycerol and dihydroxyacetone, increased the concentration of GR mRNA in primary cultures of hepatocytes from fed rats. Glucagon blunted the effect of glucose without being dominant. The stimulatory effect of those substrates was not mimicked by the nonmetabolizable carbohydrate L-glucose or the glucokinase inhibitor glucosamine or when hepatocytes were isolated from starved rats. In addition, inhibitors of gluconeogenesis and lipolysis could decrease the concentration of GR mRNA from hepatocytes of starved rats. Combined, these data strongly suggest that glucose flux in the glycolytic and gluconeogenic pathways at the level of triose intermediates could control expression of GR mRNA and participate in controlling its own metabolism.     

Suppression of glycolysis is associated with an increase in glucose cycling in hepatocytes from diabetic rats

Rates of cycling between glucose and glucose 6-phosphate and between glucose and pyruvate, and the effects of these cycles on glucose metabolism, were compared in hepatocytes isolated from fasted normal or streptozotocin-induced diabetic rats. In diabetic hepatocytes the rate of glucose phosphorylation was 30% lower than that in normal hepatocytes, and there was a doubling of the rate of glucose/glucose 6-phosphate cycling. In addition, the rate of glycolysis was 60% lower in diabetic hepatocytes. This inhibition of glycolysis and stimulation of glucose/glucose 6-phosphate cycling appeared to be a consequence of the elevated rates of endogenous fatty acid oxidation observed in diabetic hepatocytes. The proportion of glycolytically derived pyruvate that was recycled to glucose was more than doubled in hepatocytes from diabetic rats compared with normal animals. This increase also appeared to be linked to the high rates of endogenous fatty acid oxidation in diabetic cells. As a consequence of the increased rates of both these cycles, 85% of all glucose molecules taken up by diabetic hepatocytes were recycled to glucose, compared with only 50% in normal hepatocytes. Glucose cycling is therefore likely to make a substantial contribution to the hyperglycemia of diabetes.     

Red blood cell glucose metabolism in trisomy 10p: possible role of hexokinase in the erythrocyte

Red blood cell glucose metabolism was investigated in a male patient with de novo trisomy 10p. According to previous evidence, when assigning hexokinase gene locus in the 10p11 leads to pter region, a triplex dosage effect of hexokinase activity (HK) was found, while all the other erythrocyte glycolytic enzymes were in the normal values range. Red blood cell glucose utilization was 2.87 mumole/hr/ml RBC as compared to 1.43 in normal controls; the rate of glucose metabolized through the hexose monophosphate shunt (HMPS) was unchanged. Glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and dihydroxyacetone phosphate increased with respect to normal controls, while normal levels of 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP were found. The HK activity increased in all the red blood cell fractions obtained by density gradient ultracentrifugation. However, a small difference in the distribution of cells through the gradient was evident. The experiments reported in this article show that in the red blood cells of patients with trisomy 10p, an increased level of HK leads to higher concentrations of glucose-6-phosphate and to a faster glucose utilization in the Embden-Meyerhof pathway, while the HMPS rate is unchanged.