Impaired insulin secretion and decreased expression of the nutritionally responsive ribosomal kinase protein S6K-1 in pancreatic islets from malnourished rats

Low protein diet has been shown to affect the levels and activities of several enzymes from pancreatic islets. To further extend the knowledge on how malnutrition affects insulin secretion pathway, we investigated in this work the insulin release induced by glucose or leucine, an insulin secretagogue, and the expression of insulin receptor (IR), insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K), and p70S6K1 (S6K-1) proteins from pancreatic islets of rats fed a normal (17%; NP) or a low (6%; LP) protein diet for 8 weeks. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 16.7 mmol/L of glucose, or 2.8 mmol/L of glucose in the presence or absence of 20 mmol/L of leucine. Glucose- and leucine-induced insulin secretions were higher in NP than in LP islets. Western blotting analysis showed an increase in the expression of IR and PI3K protein levels whereas IRS1 and S6K-1 protein expression were lower in LP compared to NP islets. In addition, S6K-1 mRNA expression was also reduced in islets from LP rats. Our data indicate that a low protein diet modulates the levels of several proteins involved in the insulin secretion pathway. Particularly, the decrease in S6K-1 expression might be an important factor affecting either glucose- or leucine-induced insulin secretion.     

Genistein protects ovarian granulosa cells from oxidative stress via cAMP‐PKA signaling

Genistein is an isoflavone that has estrogen (E₂)‐like activity and is beneficial for follicular development, but little is known regarding its function in oxidative stress (OS)‐mediated granulosa cell (GC) injury. Here, we found that after exposure to H₂O₂, Genistein weakened the elevated levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), which were regarded as the biomarkers for OS, and rescued glutathione (GSH) content and GSH/GSSG ratio accompanying with a simultaneous increase in cyclic adenosine monophosphate (cAMP) level, whereas addition of protein kinase A (PKA) inhibitor H89 impeded the effects of Genistein on the levels of ROS and MDA. Further analysis evidenced that Genistein enhanced the activities of antioxidant enzymes superoxide dismutase (SOD), GSH‐peroxidase (GSH‐Px), and catalase (CAT) in H₂O₂‐treated GCs, but this enhancement was attenuated by H89. Under OS, Genistein improved cell viability and lessened the apoptotic rate of GCs along with a reduction in the activity of Casp3 and levels of Bax and Bad messenger RNA (mRNA), while H89 reversed the above effects. Moreover, Genistein treatment caused an obvious elevation in mitochondrial membrane potential (MMP) followed by a decline in the levels of intracellular mitochondrial superoxide, but H89 inhibited the regulation of Genistein on MMP and mitochondrial superoxide. Supplementation of Genistein promoted the secretion of E₂ and increased the expression of Star and Cyp19a1 mRNA, whereas suppressed the level of progesterone (P₄) accompanied with a decline in the level of Hsd3b1 mRNA expression. H89 blocked the regulation of Genistein on the secretion of E₂ and P₄, and alleviated the ascending of Star and Cyp19a1 elicited by Genistein. Collectively, Genistein protects GCs from OS via cAMP‐PKA signaling.     

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H₂O₂‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eM_aPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H₂O₂‐treated rat islets with increased viability (62% versus 48% H₂O₂), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

The role of old age in the effects of glucose on insulin secretion, pentosephosphate shunt activity, pyridine nucleotides and glutathione of rat pancreatic islets

In pancreatic islets of adult (three month) and old (24 month) rats the effect of glucose on glucose oxidation, pyridine nucleotides, glutathione and insulin secretion was studied. DNA content was similar in both groups of animals; however, islets of old rats exhibited 30% less insulin content. While glucose-induced (16.7 mM) insulin secretion in islets of old rats was approximately 50% less than in islets of adults, no significant difference was observed in the insulin releasing effect of theophylline (1 mM). Although islet production of 14CO2 in the presence of 16.7 mM glucose increased equally in both groups, elevation of glucose failed to increase the percentage of total glucose oxidation via the pentose phosphate shunt in islets of old rats. Elevation of glucose increased the NADPH/NADP and the NADH/NAD ratio in both groups of islets in a similar manner. The effect of glucose on the GSH/GSSG ratio revealed a dose-related increase in the islets of adult rats, whereas islets of old rats did not respond to elevation of glucose. Our data seem to indicate that the lower secretory response of islets of old rats is related to the failure of glucose to increase the GSH/GSSG ratio. In contrast the insulin release induced by theophylline does not appear to depend on islet thiols.     

Taurine supplementation restores glucose and carbachol-induced insulin secretion in islets from low-protein diet rats: involvement of Ach-M3R, Synt 1 and SNAP-25 proteins

Isolated islets from low-protein (LP) diet rats showed decreased insulin secretion in response to glucose and carbachol (Cch). Taurine (TAU) increases insulin secretion in rodent islets with a positive effect upon the cholinergic pathway. Here, we investigated the effect of TAU administration upon glucose tolerance and insulin release in rats fed on a normal protein diet (17%) without (NP) or with 2.5% of TAU in their drinking water (NPT), and LP diet fed rats (6%) without (LP) or with TAU (LPT). Glucose tolerance was found to be higher in LP, compared to NP rats. However, plasma glucose levels, during ipGTT, in LPT rats were similar to those of controls. Isolated islets from LP rats secreted less insulin in response to increasing glucose concentrations (2.8-22.2 mmol/L) and to 100 μmol/L Cch. This lower secretion was accompanied by a reduction in Cch-induced internal Ca(2+) mobilization. TAU supplementation prevents these alterations, as judged by the higher secretion induced by glucose or Cch in LPT islets. In addition, Ach-M3R, syntaxin 1 and synaptosomal associated protein of 25 kDa protein expressions in LP were lower than in NP islets. The expressions of these proteins in LPT were normalized. Finally, the sarcoendoplasmatic reticulum Ca(2+)-ATPase 3 protein expression was higher in LPT and NPT, compared with controls. In conclusion, TAU supplementation to LP rats prevented alterations in glucose tolerance as well as in insulin secretion from isolated islets. The latter effect involves the normalization of the cholinergic pathway, associated with the preservation of exocytotic proteins.     

NAD(P)H oxidase participates in the palmitate‐induced superoxide production and insulin secretion by rat pancreatic islets

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose‐stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free‐albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI—diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase‐I (CPT‐I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47^PHOX translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up‐regulated the protein content of p47^PHOX and the mRNA levels of p22^PHOX, gp91^PHOX, p47^PHOX, proinsulin and the G protein‐coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate. J. Cell. Physiol. 226: 1110–1117, 2011. © 2010 Wiley‐Liss, Inc.     

Reduced insulin secretion in response to nutrients in islets from malnourished young rats is associated with a diminished calcium uptake

Changes in (45)Ca uptake and insulin secretion in response to glucose, leucine, and arginine were measured in isolated islets derived from 4-week-old rats born of mothers maintained with normal protein (NP, 17%) or low protein (LP, 6%) diet during pregnancy and lactation. Glucose provoked a dose-dependent stimulation of insulin secretion in both groups of islets, with basal (2.8 mmol/L glucose) and maximal release (27.7 mmol/L glucose) significantly reduced in LP compared with NP islets. In the LP group the concentration-response curve to glucose was shifted to the right compared with the NP group, with the half-maximal response occurring at 16.9 and 13.3 mmol/L glucose, respectively. In LP islets, glucose-induced first and second phases of insulin secretions were drastically reduced. In addition, insulin response to individual amino acids, or in association with glucose, was also significantly reduced in the LP group compared with NP islets. Finally, in LP islets the (45)Ca uptake after 5 minutes or 90 minutes of incubation (which reflect mainly the entry and retention, respectively, of Ca(2+)), was lower than in NP islets. These data indicate that in malnourished rats both initial and sustained phases of insulin secretion in response to glucose were reduced. This poor secretory response to nutrients seems to be the consequence of an altered Ca(2+) handling by malnourished islet cells.     

Insulin secretion kinetics from single islets reveals distinct subpopulations

Type II diabetes progresses with inadequate insulin secretion and prolonged elevated circulating glucose levels. Also, pancreatic islets isolated for transplantation or tissue engineering can be exposed to glucose over extended timeframe. We hypothesized that isolated pancreatic islets can secrete insulin over a prolonged period of time when incubated in glucose solution and that not all islets release insulin in unison. Insulin secretion kinetics was examined and modeled from single mouse islets in response to chronic glucose exposure (2.8‐20 mM). Results with single islets were compared to those from pools of islets. Kinetic analysis of 58 single islets over 72 h in response to elevated glucose revealed distinct insulin secretion profiles: slow‐, fast‐, and constant‐rate secretors, with slow‐secretors being most prominent (ca., 50%). Variations in the temporal response to glucose therefore exist. During short‐term (<4 h) exposure to elevated glucose few islets are responding with sustained insulin release. The model allowed studying the influence of islet size, revealing no clear effect. At high‐glucose concentrations, when secretion is normalized to islet volume, the tendency is that smaller islets secrete more insulin. At high‐glucose concentrations, insulin secretion from single islets is representative of islet populations, while under low‐glucose conditions pooled islets did not behave as single ones. The characterization of insulin secretion over prolonged periods complements studies on insulin secretion performed over short timeframe. Further investigation of these differences in secretion profiles may resolve open‐ended questions on pre‐diabetic conditions and transplanted islets performance. This study deliberates the importance of size of islets in insulin secretion. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1059–1068, 2018     

Protein deficiency attenuates the effects of alloxan on insulin secretion and glucose homeostasis in rats.

We have investigated the effect of alloxan on insulin secretion and glucose homeostasis in rats maintained on a 17% protein (normal protein, NP) or 6% protein (low protein, LP) diet from weaning (21 days old) to adulthood (90 days old). The incidence of alloxan diabetes was higher in the NP (3.5 times) than in the LP group. During an oral glucose tolerance test, the area under serum glucose curve was lower in LP (57%) than in NP rats while there were no differences between the two groups in the area under serum insulin curve. The serum glucose disappearance rate (Kitt) after exogenous insulin administration was higher in LP (50%) than in NP rats. In pancreatic islets isolated from rats not injected with alloxan, acute exposure to alloxan (0.05 mmol/L) reduced the glucose- or arginine-stimulated insulin secretion of NP islets by 78% and 56%, respectively, whereas for islets from LP rats, the reduction was 47% and 17% in the presence of glucose and arginine, respectively. Alloxan treatment reduced the glucose oxidation in islets from LP rats to a lesser extent than in NP islets (23% vs. 56%). In conclusion, alloxan was less effective in producing hyperglycemia in rats fed a low protein diet than in normal diet rats. This effect is attributable to an increased peripheral sensivity to insulin in addition to a better preservation of glucose oxidation and insulin secretion in islets from rats fed a low protein diet.     

N‐acetyl‐L‐cysteine inhibits bleomycin induced apoptosis in malignant testicular germ cell tumors

Antioxidants may prevent apoptosis of cancer cells via inhibiting reactive oxygen species (ROS). However, to date no study has been carried out to elucidate the effects of strong antioxidant N‐acetylcysteine (NAC) on Bleomycin induced apoptosis in human testicular cancer (NTERA‐2, NT2) cells. For this reason, we studied the effects of Bleomycin and NAC alone and in combination on apoptotic signaling pathways in NT2 cell line. We determined the cytotoxic effect of bleomycin on NT2 cells and measured apoptosis markers such as Caspase‐3, ‐8, ‐9 activities and Bcl‐2, Bax, Cyt‐c, Annexin V‐FTIC and PI levels in NT2 cells incubated with different agents for 24 h. Early apoptosis was determined using FACS assay. We found half of the lethal dose (LD₅₀) of Bleomycin on NT2 cell viability as 400, 100, and 20 µg/ml after incubations for 24, 48, and 72 h, respectively. Incubation with bleomycin (LD₅₀) and H₂O₂ for 24 h increased Caspase‐3, ‐8, ‐9 activities, Cyt‐c and Bax levels and decreased Bcl‐2 levels. The concurrent incubation of NT2 cells with bleomycin/H₂O₂ and NAC (5 mM) for 24 h abolished bleomycin/H₂O₂‐dependent increases in Caspase‐3, ‐8, ‐9 activities, Bax and Cyt‐c levels and bleomycin/H₂O₂‐dependent decrease in Bcl‐2 level. Our results indicate that bleomycin/H₂O₂ induce apoptosis in NT2 cells by activating mitochondrial pathway of apoptosis, while NAC diminishes bleomycin/H₂O₂ induced apoptosis. We conclude that NAC has antagonistic effects on Bleomycin‐induced apoptosis in NT2 cells and causes resistance to apoptosis which is not a desired effect in eliminating cancer cells. J. Cell. Biochem. 114: 1685–1694, 2013. © 2013 Wiley Periodicals, Inc.     

High level of reduced glutathione contributes to detoxification of lipid peroxide‐derived reactive carbonyl species in transgenic Arabidopsis overexpressing glutathione reductase under aluminum stress

Lipid peroxide‐derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage‐inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR‐OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild‐type under Al stress. Exogenous application of 4‐hydroxy‐2‐nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild‐type plants. GR‐OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H₂O₂ due to Al stress in GR‐OE plants was lower by 26% than in wild‐type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)‐2‐heptenal and n‐octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Increased L-CPT-1 activity and altered gene expression in pancreatic islets of malnourished adult rats: a possible relationship between elevated free fatty acid levels and impaired insulin secretion

Intrauterine growth restriction is associated with chronically elevated levels of serum fatty acids and reduced glucose-stimulated insulin secretion. Lipid metabolism in pancreatic beta cells is critical for the regulation of insulin secretion, and the chronic exposure to fatty acids results in higher palmitate oxidation rates and an altered insulin response to glucose. Using a rat model of isocaloric protein restriction, we examined whether pre- and postnatal protein malnutrition influences the properties of pancreatic islet carnitine palmitoyltransferase-1 (liver isoform, L-CPT-1), a rate-limiting enzyme that regulates fatty acid oxidation in mitochondria. The activity of L-CPT-1 in pancreatic islets increased in the low protein (LP), although the L-CPT-1 mRNA levels were unaffected by malnutrition. The susceptibility of enzyme to inhibition by malonyl-CoA was unaltered and the content of malonyl-CoA was reduced in LP cells. Because the mitochondrial oxidation of fatty acids is related to the altered expression of a number of genes encoding proteins involved in insulin secretion, the levels of expression of insulin and GLUT-2 mRNA were assessed. A reduced expression of both genes was observed in malnourished rats. These results provide further evidence that increased L-CPT-1 activity and changes in gene expression in pancreatic islets may be involved in the reduced insulin secretion seen in malnourished rats.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

Redox changes during cold acclimation affect freezing tolerance but not the vegetative/reproductive transition of the shoot apex in wheat

Cold acclimation is necessary for winter wheat (Triticum aestivum L.) to achieve its genetically determined maximum freezing tolerance, and cold also fulfils the vernalisation requirement. Chromosome 5A is a major regulator of these traits. The aim of the present study was to discover whether changes in the half‐cell redox potential of the glutathione/glutathione disulphide (GSH/GSSG) and ascorbate/dehydroascorbate (AA/DHA) couples induced by cold acclimation are related to freezing tolerance and vernalisation requirement in a specific genetic system including chromosome 5A substitution lines. The amounts of H₂O₂ and AA, and the AA/DHA ratio showed a rapid and transient increase in the crown of all genotypes during the first week of acclimation, followed by a gradual increase during the subsequent 2 weeks. The amount of GSH and its ratio compared to GSSG quickly decreased during the first day, while later these parameters showed a continuous slow increase. The H₂O₂, AA and GSH concentrations, AA/DHA and GSH/GSSG ratios and the half‐cell reduction potential of the GSH/GSSG couple were correlated with the level of freezing tolerance after 22 days at 2 °C; hence these parameters may have an important role in the acclimation process. In contrast to H₂O₂ and the non‐enzymatic antioxidants, the lipid peroxide concentration and activity of the four antioxidant enzymes exhibited a transient increase during the first week, with no significant difference between genotypes. None of the parameters studied showed any relationship with the vegetative/generative transition state monitored as apex morphology and vernalisation gene expression.     

Increased resistance to hydrogen peroxide‐induced cardiac contracture is associated with decreased myocardial oxidative stress in hypothyroid rats

The purpose of this study was to determine whether decreased oxidative stress would increase the resistance to cardiac contracture induced by H₂O₂ in hypothyroid rats. Male Wistar rats were divided into two groups: control and hypothyroid. Hypothyroidism was induced via thyroidectomy. Four weeks post surgery, blood samples were collected to perform thyroid hormone assessments, and excised hearts were perfused at a constant flow with or without H₂O₂ (1 mmol/L), being divided into two sub‐groups: control, hypothyroid, control + H₂O₂, hypothyroid + H₂O₂. Lipid peroxidation (LPO) was evaluated by chemiluminescence (CL) and thiobarbituric acid reactive substances (TBARS) methods, and protein oxidation by carbonyls assay in heart homogenates. Cardiac tissue was also screened for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, and for total radical‐trapping antioxidant potential (TRAP). Analyses of SOD and glutathione‐S‐transferase (GST) protein expression were also performed in heart homogenates. Hypothyroid hearts were found to be more resistant to H₂O₂‐induced contracture (60% elevation in LVEDP) as compared to control. CL, TBARS, carbonyl, as well as SOD, CAT, GPx activities and TRAP levels were reduced (35, 30, 40, 30, 16, 25, and 33%, respectively) in the cardiac homogenates of the hypothyroid group as compared to controls. A decrease in SOD and GST protein levels by 20 and 16%, respectively, was also observed in the hypothyroid group. These results suggest that a hypometabolic state caused by thyroid hormone deficiency can lead to an improved response to H₂O₂ challenge and is associated with decreased oxidative myocardial damage. Copyright © 2009 John Wiley & Sons, Ltd.     

Metabolic Adaptations to Dexamethasone‐Induced Insulin Resistance in Healthy Volunteers

Objective : Insulin resistance is observed in individuals with normal glucose tolerance. This indicates that increased insulin secretion can compensate for insulin resistance and that additional defects are involved in impaired glucose tolerance or type 2 diabetes. The objective of this study was to evaluate a procedure aimed at assessing the compensatory mechanisms to insulin resistance. Research Methods and Procedures : Eight healthy nonobese female patients were studied on two occasions, before and after administration of 2 mg/d dexamethasone for 2 days during a two‐step hyperglycemic clamp. Insulin secretion was assessed from plasma insulin concentrations. Insulin sensitivity was assessed from the ratio of whole‐body glucose use (6, 6 ²H₂ glucose) to plasma insulin concentrations. This procedure is known to induce a reversible impairment of glucose tolerance and insulin resistance. Results : In all subjects, dexamethasone induced a decrease in insulin sensitivity and a proportionate increase in first‐phase insulin secretion and in insulin concentrations at both steps of glycemia. The resulting hyperinsulinemia allowed the restoration of normal whole‐body glucose uptake and the suppression of plasma free fatty acids and triglycerides. In contrast, the suppression of endogenous glucose production was impaired after dexamethasone (p < 0.01). Discussion : Increased insulin secretion fully compensates dexamethasone‐induced insulin resistance in skeletal muscle and adipose tissue but not in the liver. This suggests that failure to overcome hepatic insulin resistance can impair glucose tolerance. The compensatory insulin secretion in response to insulin resistance can be assessed by means of a hyperglycemic clamp after a dexamethasone challenge.