A study of the effect of oral glucose loading on plasma oxidant:antioxidant balance in normal subjects

Summary Background Antioxidant defence has been reported to decrease, and oxidative stress to increase, after oral glucose loading in both normal and diabetic subjects. If confirmed in normal subjects, glucose–induced antioxidant depletion has important implications for health in relation to the modern, sugar–rich diet. Aim of the study To investigate changes in plasma biomarkers of oxidant:antioxidant balance in non–diabetic subjects following oral glucose loading. Baseline inter–relationships between biomarkers of glycaemic control, oxidant:antioxidant balance and inflammation were also explored. Methods A singleblinded, placebo–controlled, crossover intervention trial involving 10 healthy, consenting subjects. Venous blood was collected after ingestion of 75 g glucose in 300 mL water, or of water alone. Blood was collected at 0 time (fasting) and 30, 60, 90, 120 min post–ingestion. Within 2 weeks the procedure was repeated with volunteers crossedover onto the other treatment. Plasma total antioxidant capacity (as the FRAP value), ascorbic acid, –tocopherol, uric acid, malondialdehyde (MDA), allantoin and high sensitivity C–reactive protein (hsCRP), glucose and insulin, were measured in all samples. Paired results post–glucose and post–water at each time interval were compared using the Wilcoxon matched–pairs signed–ranks test. Results Normal glucose tolerance was observed in all subjects, although, as expected, plasma glucose and insulin increased significantly (p < 0.05, n = 10) after glucose loading. Post–glucose responses in plasma FRAP and the individual antioxidants tested were not significantly different to the responses seen post–water, although both FRAP and –tocopherol decreased slightly. Neither were postglucose changes in plasma MDA and allantoin, putative biomarkers of oxidative stress, significantly different to those after intake of water alone. Plasma FRAP and -tocopherol also decreased slightly, but not significantly, after intake of water. A significant direct correlation (r = 0.867, p < 0.001, n = 10) was found between fasting allantoin and (log transformed) hsCRP concentrations. Conclusions These new data from a controlled intervention trial indicate that acute, transient increases in plasma glucose following oral intake of a large glucose load do not, as previously reported, cause a significant decrease in plasma antioxidants or increase oxidative stress in non–diabetic subjects. This is reassuring given the large quantities of sugar ingested by children and adolescents. However, a small decrease in plasma antioxidant capacity was seen after ingestion of water and of glucose, and it is possible that intake of glucose without concomitant intake of antioxidants in susceptible individuals may cause oxidative stress. Further work is needed in relation to diabetic subjects and a possible glucose threshold for this. The finding of a direct relationship between allantoin, a biomarker of oxidative stress, and hsCRP, a marker of inflammation and CHD predictor, in healthy subjects is interesting and indicates a link between sub–clinical inflammation and oxidative stress.     

Plasma total homocysteine and gallstone in middle-aged Japanese men

Background We wished to determine whether selected coronary risk factors correlated with the presence of gallstone (GS) in middle-aged Japanese men. Methods We carried out a cross-sectional study of 974 male Self-Defense officials. The odds ratio of a 1-SD increment in levels of coronary risk factors for the presence of GS was calculated in a logistic regression analysis. Results Nine subjects had undergone cholecystectomy. Gallstone was detected in 39 subjects and not in 926 subjects. The presence of GS was associated with total homocysteine (tHcy), but not with total cholesterol, triglyceride, γ-glutamyl transferase, glucose, or folate. The odds ratio of a 2.8-μmol/l increase (1 SD) in tHcy levels for the presence of GS was 1.34 (95% confidence interval [CI], 1.05–1.69; P = 0.017), and the ratio was 1.33 (95% CI, 1.04–1.70; P = 0.025) after being adjusted for lifestyle factors, including cigarette smoking, vegetable intake, physical activity, and abstention from alcohol. Conclusions Plasma tHcy, but not the other coronary risk factors, correlated with the presence of GS in middle-aged Japanese men. This association may partly explain the reported high prevalence rate of coronary heart disease in subjects with GS.     

Oxidative metabolic responses with recurrent seizures in rat cerebral cortex: Role of systemic factors

Simultaneous focal measurements of cerebral oxygen tension and cytochrome a,a3 redox levels were made in rat cortex in order to obtain a direct and continuous assessment of oxidative metabolic changes during serial seizures. Initial seizures evoked by pentylenetetrazol were accompanied by transient increases in tissue pO2 and cytochrome a,a3 oxidation, confirming that oxygen provision is adequate to meet metabolic demand. After some point, subsequent seizures were accompanied by failure of pO2 to increase and failure of cytochrome a,a3 to oxidize, or by decreases in tissue pO2 and shifts in the redox level of cytochrome a,a3 toward reduction, signalling cortical oxygen insufficiency. Whereas early seizures were accompanied by increments in both cerebral blood volume and arterial blood pressure, one or both of these variables failed to increment during later seizures. This was particularly evident following the onset of spontaneously recurring seizures with short intervals between bursts of ECoG activity. These investigations emphasize the importance of systemic factors in determining the cerebral metabolic response to seizures and support the suggestion that neuronal damage in status epilepticus may be the result of derangements of oxidative metabolism.     

Experiential input alters the phosphorylation of specific proteins in brain membranes

The effects of a training experience that involves foot shock on the endogenous phosphorylation of membrane-bound proteins from brain were studied. Crude membrane fractions were prepared from the cerebral cortex and neostriatum of animals that had been sacrificed by quick freezing. In vitro incubation of the membranes with gamma-^{3 2}P-ATP, followed by SDS-gel electrophoresis of the phosphorylated substrates, revealed that the phosphorylation of two protein components (designated F and H-I) increased in preparations from animals that were subjected to a training experience 24 hr prior to sacrifice. These effects were greater in preparations from the neostriatum than from the cerebral cortex, and were observed in experiments using both rats and mice. Although all trained animals showed a high phosphorylation of bands F and H-I, control animals showed a greater variability in the phosphorylation of these bands. The results indicate that the phosphorylation of specific proteins may play a mediatory role in the processing of experiential information.     

INFLUENCE OF DIETARY PROTEIN AND CARBOHYDRATE ON OXIDATIVE BIOTRANSFORMATION OF DRUGS IN NORMAL ADULTS AND CHILDREN WITH ASTHMA

The macronutrient composition of the diet has been shown to influence the oxidative biotransformation of two prototype drugs, antipyrine and theophylline, in both normal adults and asthmatic children.     

The effect of myelin basic protein on the endogenous phosphorylation of platelets

Bovine myelin basic protein (BP) induced a shape change and endogenous phosphorylation of a 45,000 (45K) molecular weight protein of intact human platelets. This effect occurred rapidly over an effective concentration range of 5–100 uM BP. BP peptides encompassing residues 1–42, 43–88, and 89–169 from the BP molecule of 169 residues, neither induced phosphorylation of platelets nor blocked the effect of intact BP. Subfractionation of disrupted platelets demonstrated the phosphorylated 45K protein in the 100,000 xg supernate. When isolated platelet membranes were used. no BP induced phosphorylation of a 45K protein could be detected. The amino acid composition of the purified, phosphorylated 45K protein differed from those of other known platelet proteins. BP itself was also phosphorylated by an endogenous platelet protein kinase(s) present both in the 100,000 xg supernatant and in the isolated membrane fraction of platelets. These results indicate that the normal or pathological release of BP from myelin may lead to phosphorylation of an internal protein of platelets and possibly other tissue elements with resultant metabolic and functional changes.     

Expression of KIT, EGFR, HER-2 and tyrosine phosphorylation in undifferentiated thyroid carcinoma: implication for a new therapeutic approach

The KIT, epidermal growth factor receptor (EGFR) and HER-2 oncoproteins have tyrosine kinase activity and are molecular targets in human cancer therapy. To clarify the significance of KIT, EGFR, and HER-2 in undifferentiated thyroid carcinoma (UTC), the expression of these receptors and tyrosine phosphorylation was examined immunohistochemically in resected cases of UTC and papillary thyroid carcinoma (PTC). KIT, EGFR, and HER-2 were also examined at the protein and mRNA levels in five UTC cell lines. KIT expression (1+), EGFR overexpression (2+/3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunohistochemically in 40%, 70%, 10%, and 50% of the 10 UTC. In 20 PTC, KIT, EGFR, and HER-2 were not detected, but tyrosine phosphorylation was detected in 25% of cases. In the five UTC cell lines, KIT expression (1+), EGFR overexpression (3+), HER-2 expression (1+), and tyrosine phosphorylation were detected immunocytochemically in 60%, 100%, 20%, and 40%, respectively. Western blot analysis did not detect KIT expression, but did detect EGFR and HER-2 expression in all five cell lines. Real-time polymerase chain reaction detected KIT mRNA in two of the cell lines (40%), EGFR in five (100%), and HER-2 in three (60%). The present findings suggest that EGFR overexpression was involved in the proliferation and development of UTC and was frequently accompanied by tyrosine phosphorylation. Expression of KIT and HER-2 appeared to be weak but significant, suggesting a possible role in the development of UTC. Molecular therapies targeting KIT, EGFR, HER-2, and/or tyrosine phosphorylation might be indicated for UTC.     

Apoptotic cell death progression after photothrombotic focal cerebral ischaemia: effects of the lipophilic iron chelator 2,2'-dipyridyl

Two different forms of cell death have been distinguished morphologically following cerebral ischaemia: necrotic and apoptotic cell death. The aim of this study was to investigate the contribution of apoptosis to ischaemic damage by carefully depicting the temporal and spatial neuronal death following focal ischaemia. For this purpose, rats were subjected to chemical photothrombosis, and histological and biochemical analyses were performed over a period of 24 h after the onset of ischaemia. In addition, the effects of the lipophilic antioxidant iron chelator 2,2'-dipyridyl (DP) were evaluated 24 h after photothrombosis when the lesion volume was maximal. Our results showed two separate waves of neuronal death. In the first wave, shrunken dark neurons were massively present as early as 2 h after photothrombosis in the infarct core. From this initial neuronal abnormal population, progressive and time-dependent changes of both necrotic and apoptotic cell death were observed, leading to ghost neurons and apoptotic bodies after 24 h. The extension of the lesion coincided with a second wave of cell death. Massive and rapid neuronal loss occurred at the infarct border, which appeared as a sharply demarcated pale region. Procaspase and poly(ADP-ribose) polymerase-1 (PARP-1) cleavages were also detected in the infarct core and surrounding damaged tissue. DP treatment markedly blocked the enlargement of the lesion, the infarct border being rescued from infarction. Furthermore, a large decrease of apoptotic bodies was associated with a significant drop of caspase and PARP-1 cleavages, suggesting that the protective effect of DP closely correlates with limitation of apoptosis expansion.     

Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices

A hallmark in the development of GABAergic neurotransmission is the switch in GABA(A)-mediated responses from depolarizing to hyperpolarizing. This occurs due to a gradual decrease in the intracellular concentration of chloride caused by the functional expression of the neuron-specific K-Cl cotransporter KCC2. Whether a mere increase in the amount of KCC2 protein is the rate-limiting step in vivo, or a further activation of the otherwise nonfunctional cotransporter is required, is not clear. Imposing a fixed Cl(-) load via patch pipette we measured the resultant somato-dendritic gradients in reversal potential of GABAergic currents to determine the time course of functional maturation of KCC2-mediated Cl(-) extrusion in two preparations: cultured mouse hippocampal neurons plated at embryonic day 17 and CA1 pyramidal cells in acute slices. We found that in immature neurons in both preparations the gradient is initially small or not detectable. It undergoes an abrupt increase at around days 13-14 in culture, while a more gradual increase occurs between postnatal days 5-14 in slices. Consistent with the presence of a nonfunctional form of KCC2 in immature hippocampal neurons grown in culture, application of the broad-spectrum kinase inhibitor staurosporine produces a rapid and potent up-regulation of KCC2 function in these cultured neurons, but not in neonatal slices. Taken together with our previously published data, these results indicate that the functional activity of KCC2 in vivo parallels the developmental expression of the protein, whereas cultured neurons require an additional activation step (mimicked by staurosporine) for KCC2 to become functional.     

Toxin-based selection of insulin-producing cells with improved defense properties for islet cell transplantation

Insulin-producing pancreatic beta-cells are known to be extremely susceptible to destruction, primarily by autoimmune mechanisms, infectious agents, and by chemical toxins that cause overt type I diabetes. As development of highly protected insulin-producing cells would be important for successful cell therapy of diabetic patients, gene transfection technique was utilized by several investigators in order to improve the defense properties of transplanted cells. In this article, we summarize other approaches based on a selection strategy that has been developed in our laboratory and by other research groups that engineer pancreatic beta-cells to provide protection against diabetogenic toxins (streptozotocin and alloxan), oxidative stress and cytokines. Selection strategies based on acute repeated or long-term continuous treatment of cell lines with cytotoxic agents have resulted in the selection of highly resistant cell subpopulations. We discuss possible involvement of different expression of cytoprotective genes in the selection of cell subpopulations, which demonstrate a broad spectrum of resistance. Importantly, toxin-based selection did not impair functional activity of the cells as it was shown in vitro. In addition, selected cells preserved their improved metabolic characteristics following encapsulation in alginate and subsequent implantation in diabetic animals. Identifying the mechanisms through which cell defense properties act will help clarify the process responsible for beta-cell regeneration in type I diabetes patients. Such knowledge might be useful in developing strategies focusing on the regeneration of beta-cell resistant populations.