Nerve glucose, fructose, sorbitol, myo-inositol, and fiber degeneration and regeneration in diabetic neuropathy

We measured the alcohol sugars in sural nerves from 11 controls, 21 conventionally treated patients with diabetes and neuropathy, and 4 diabetics without neuropathy. The results were related to metabolic control and to clinical, neuropathological, and morphometric abnormalities in the nerves. The mean endoneurial glucose, fructose, and sorbitol values were higher in diabetic patients than in controls. Linear regression analysis revealed that nerve sorbitol content in the diabetics was inversely related to the number of myelinated fibers (P = 0.003). Mean nerve levels of myo-inositol were not decreased in the diabetic patients, with or without neuropathy, and were not associated with any of the neuropathological end points of diabetes. Our results indicate that myo-inositol deficiency is not part of the pathogenesis of human diabetic neuropathy, as had been hypothesized. Other accumulated alcohol sugars, however, were increased in diabetes and were associated with the severity of neuropathy. On repeat biopsy, six diabetics, treated for a year with the aldose reductase inhibitor sorbinil, had decreased endoneurial levels of sorbitol (P less than 0.01) and fructose (0.05 less than P less than 0.1), but unchanged levels of myo-inositol.     

Iron depletion by deferoxamine up-regulates glucose uptake and insulin signaling in hepatoma cells and in rat liver

Iron depletion improves insulin resistance in patients with nonalcoholic fatty liver disease and diabetes and also stabilizes the hypoxia-inducible factor (HIF)-1, resulting in increased glucose uptake in vitro. This study investigated the effect of iron depletion by deferoxamine on insulin signaling and glucose uptake in HepG2 hepatocytes and in rat liver. In HepG2 cells, deferoxamine stabilized HIF-1alpha and induced the constitutive glucose transporter Glut1 and the insulin receptor. Up-regulation of insulin receptor by deferoxamine was mimicked by the intracellular iron chelator deferasirox and the hypoxia inducer CoCl2 and required the HIF-1 obligate partner ARNT/HIF-1beta. Iron depletion increased insulin receptor activity, whereas iron supplementation had the opposite effect. Deferoxamine consistently increased the phosphorylation status of Akt/PKB and its targets FoxO1 and Gsk3beta, which mediate the effect of insulin on gluconeogenesis and glycogen synthesis, and up-regulated genes involved in glucose uptake and utilization. Iron depletion of Sprague-Dawley rats increased HIF-1alpha expression, improved glucose clearance, and was associated with up-regulation of insulin receptor and Akt/PKB levels and of glucose transport in hepatic tissue. Conversely, gluconeogenic genes were not affected. In rats with fatty liver because of a high-calorie and high-fat diet, glucose clearance was increased by iron depletion and decreased by iron supplementation. Thus, iron depletion by deferoxamine up-regulates glucose uptake, and increases insulin receptor activity and signaling in hepatocytes in vitro and in vivo.     

Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms

Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.     

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.     

Correlation between glucose uptake and membrane potential in Leishmania parasites isolated from DCL patients with therapeutic failure: a proof of concept

Besides infection with drug-resistant parasites, therapeutic failure in leishmaniasis may be caused by altered drug pharmacokinetics, re-infection, and host immunologic compromise. Our aim has been to evaluate if relapses that occur in patients suffering from diffuse cutaneous leishmaniasis (DCL) associate with changes in the fitness of infecting organisms. Therefore, in isolates from patients suffering DCL, we correlated glucose uptake and plasma membrane potential and compared the results with those obtained from reference strains. The data demonstrate that Leishmania parasites causing DCL incorporate glucose at an efficient rate, albeit without significant changes in the plasma membrane potential as their corresponding reference strains. The isolate that did not change its accumulation rate of glucose compared to its reference strain expressed a less polarized membrane potential that was insensitive to mitochondrial inhibitors, suggesting a metabolic dysfunction that may result in glycolysis being the main source of ATP. The results constitute a proof of concept that indicates that parasites causing DCL adapted well to drug pressure and expressed an increased fitness. That is, that in Leishmania mexicana and Leishmania amazonensis, parasites isolated from DCL patients, a strong modification of the parasite physiology might occur. As consequences, the parasites adapted well to drug pressure, increased their fitness, and they had an efficient glucose uptake rate albeit not significant changes in membrane potential as their corresponding reference strains. Further validation of the concepts herein established and whether or not the third isolate corresponds with a drug-resistant phenotype need to be demonstrated at the genetic level.     

Stage-specific development of a novel adenosine transporter in Leishmania donovani amastigotes

Leishmania donovani, like all other kinetoplastida, is a purine auxotroph. Comparative studies of adenosine transport in L. donovani amastigotes and promastigotes revealed that, unlike the promastigote stage, the amastigote possesses two distinct adenosine transporters (T(1) and T(2)) both with high affinities (K(m), 1.14+/-0.05 and 2. 09+/-0.13 microM, respectively). One of these transporters (T(1)) appears to be identical with the adenosine/pyrimidine nucleoside transporter of the promastigote reported earlier. The other transporter (T(2)) is specific for the amastigote stage and transports only purine nucleosides. The biological significance of this stage-specific development of the second adenosine transporter has been briefly discussed.     

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.     

Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos

The expression of glucose transporters 1, 2, 3 and 4 was evaluated in human oocytes and polyploid preimplantation embryos. Only glucose transporter 1 (GLUT-1) isoform was detected in oocytes and in 2-12-cell stage embryos. Glucose uptake was markedly increased in embryos as compared to oocytes (19.7 +/- 3.4 pmol/min/embryo and 2.3 +/- 0.3 pmol/min/oocyte), and GLUT-1 was inhibited by cytochalasin B. These results suggest that, although GLUT-1 is expressed in human oocytes and throughout preimplantation development, its function in mediating the rise in glucose uptake is triggered following fertilization.      

Effects of adenosine uptake blockers and adenosine on evoked potentials of guinea-pig olfactory cortex

The olfactory cortex slice preparation from guinea-pig has been used to test compounds which inhibit the cellular uptake of adenosine. The uptake inhibitors dipyridamole (0.1-10 mumol/l), dilazep (1-10 mumol/l) nitrobenzylthioguanosine (1-10 mumol/l), nitrobenzylthioinosine (0.1-5 mumol/l), and hexobendine (1-100 mumol/l) increased the potency of adenosine (0.1-30 mumol/l) by up to 5-fold but did not potentiate cyclohexyladenosine (0.01-10 mumol/l). The benzodiazepine, diazepam (1 mumol/l) slightly increased the potency of adenosine (by 1.7-fold) whereas flurazepam (3 mumol/l) had no effect, suggesting that inhibition of adenosine uptake is probably not the major therapeutic action of these compounds. The uptake inhibitors depressed the amplitude of the monosynaptic epsp when added alone, an effect reversed by adenosine deaminase (1 unit/ml) whereas the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (10 mumol/l) had no effect on adenosine action. These results show that in this preparation (a) adenosine action is attenuated by an uptake mechanism and (b) endogenous adenosine release normally has no apparent effects on synaptic transmission at low stimulus rates. Nitrobenzylthioinosine and nitrobenzylthioguanosine are probably the best uptake blockers.     

The hepatocellular uptake of glucose, galactose and fructose in conscious sheep.

1. Surgical techniques for chronic catheterization of hepatic and portal veins in the sheep are described. These catheters remained usable for 2-6 months and did not alter hepatic morphology. 2. Hepatocellular uptake of monosaccharides was estimated from their ability to pass the boundaries of the sucrose space in a double indicator dilution procedure in conscious fed sheep. 3. A large proportion (81%) of D-glucose carried in the portal blood was found to enter an hepatic cellular compartment. 4. The radioactive label of D-glucose infused in the portal vein remained associated with D-glucose in hepatic venous blood samples during the experimental period. 5. A large proportion (74%) of an infused trace of D-galactose, a smaller proportion (33%) of D-fructose, and negligible amounts of L-glucose were taken up in a single passage through the liver. 6. Raised blood concentrations of sucrose or of methyl-alpha-D-glucoside (Me-alpha-DG) significantly diminished the proportional uptake of D-glucose. Raised blood concentrations of glucose, galactose or Me-alpha-DG diminished the proportional uptake of D-galactose. Raised blood concentrations of fructose diminished the proportional uptake of fructose. 7. Neither total hepatic blood flow changes nor competitive effects within the cell could account for these findings. 8. It is concluded that these monosaccharides enter the liver cell by facilitated diffusion, and share at least some of the membrane elements that mediate this process. It seems likely that only a proportion of the glucose-transporting apparatus is accessible to galactose.     

Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).     

AMP-activated protein kinase and muscle glucose uptake

The AMP-activated protein kinase (AMPK) is an enzyme that is activated in situations where there are changes in the cellular energy status such as muscle contraction and hypoxia. AMPK can also be pharmacologically activated by the compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the antidiabetic agent metformin. Several studies support the hypothesis that AMPK plays an important role in the stimulation of muscle glucose uptake by these physiological and pharmacological stimuli. In isolated rat muscles, activation of AMPK is associated with increases in glucose uptake through an insulin-independent mechanism. Studies done in rodents have shown that the activation of AMPK by AICAR is accompanied by decreases in blood glucose concentrations, in part due to enhanced muscle glucose uptake. Similar to exercise, AICAR not only directly stimulates glucose uptake into the skeletal muscle, but also enhances insulin sensitivity. The activation of AMPK and associated increases in muscle glucose uptake are affected by factors such as glycogen content, exercise training and fibre type. The effects of AMPK on muscle glucose uptake makes this protein a promising pharmacological target for the treatment of type 2 diabetes.     

胰岛素促进狗缺血心肌葡萄糖转运子1移位和葡萄糖摄取

观察胰岛素能否刺激缺血心肌葡萄糖转运子 1(GLUT1)移位和葡萄糖摄取。利用自动分析仪测定生理代谢参数 ,应用免疫印迹和免疫荧光法检测GLUT1。胰岛素使缺血心肌细胞质膜GLUT1明显增加 (从 5 2 %± 4%～ 6 7%± 6 % ,P <0 0 5 )。细胞器膜GLUT1则相应减少 ,同时伴随葡萄糖摄取量明显增加 ,是单纯缺血心肌葡萄糖摄取量的 2倍。胰岛素刺激引起GLUT1移位 ,使缺血心肌葡萄糖摄取增加。提示心肌缺血时 ,应用胰岛素有助于增加心肌葡萄糖的摄取和利用     

The tuberous sclerosis complex regulates trafficking of glucose transporters and glucose uptake

Human cancers often display an avidity for glucose, a feature that is exploited in clinical staging and response monitoring by using (18)F-fluoro-deoxyglucose (FDG) positron emission tomography. Determinants of FDG accumulation include tumor blood flow, glucose transport, and glycolytic rate, but the underlying molecular mechanisms are incompletely understood. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin complex (mTORC) 1 pathway has been implicated in this process via the hypoxia-inducible factor alpha-dependent expression of vascular endothelial growth factor and glycolytic enzymes. Thus, we predicted that tumors with elevated mTORC1 activity would be accompanied by high FDG uptake. We tested this hypothesis in eight renal angiomyolipomas in which the loss of tuberous sclerosis complex (TSC) 1/2 function gave rise to constitutive mTORC1 activation. Surprisingly, these tumors displayed low FDG uptake on positron emission tomography. Exploring the underlying mechanisms in vitro revealed that Tsc2 regulates the membrane localization of the glucose transporter proteins (Glut)1, Glut2, and Glut4, and, therefore, glucose uptake. Down-regulation of cytoplasmic linker protein 170, an mTOR effector, rescued Glut4 trafficking in Tsc2(-/-) cells, whereas up-regulation of Akt activity in these cells was insufficient to redistribute Glut4 to the plasma membrane. The effect of mTORC1 on glucose uptake was confirmed using a liver-specific Tsc1- deletion mouse model in which FDG uptake was reduced in the livers of mutant mice compared with wild-type controls. Together, these data show that mTORC1 activity is insufficient for increased glycolysis in tumors and that constitutive mTOR activity negatively regulates glucose transporter trafficking.