Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. IP6Ks convert IP6 to pyrophosphates such as diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. The inositol hexakisphosphate kinase 2 (IP6K2) controls cellular apoptosis. To explore roles for IP6K2 in brain function, we elucidated its protein interactome in mouse brain revealing a robust association of IP6K2 with creatine kinase-B (CK-B), a key enzyme in energy homeostasis. Cerebella of IP6K2-deleted mice (IP6K2-knockout [KO]) produced less phosphocreatine and ATP and generated higher levels of reactive oxygen species and protein oxidative damage. In IP6K2-KO mice, mitochondrial dysfunction was associated with impaired expression of the cytochrome-c1 subunit of complex III of the electron transport chain. We reversed some of these effects by combined treatment with N-acetylcysteine and phosphocreatine. These findings establish a role for IP6K2–CK-B interaction in energy homeostasis associated with neuroprotection.

Inositol pyrophosphates are versatile messenger molecules that mediate a variety of cellular functions, including cell growth, apoptosis, endocytosis, and cell differentiation. The most extensively studied inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), displays a 5′-diphosphate (1, 2). IP7 is generated in mammals by a family of inositol hexakisphosphate kinases (IP6Ks) (3, 4). IP6Ks exists in three isoforms: IP6K1, IP6K2, and IP6K3. Inositol hexakisphosphate kinase-2 (IP6K2) sensitizes cells to apoptosis (5, 6). Mice with targeted deletion of IP6K2 display an increased incidence of aero-digestive tract carcinoma (7). Cell survival associated with heat shock protein 90 also involves IP6K2 (8, 9).We previously reported a major role for IP6K2 in the disposition of cerebellar granule cells as well as Purkinje cell morphology and motor coordination. The influence of IP6K2 upon cerebellar disposition involved protein 4.1N, both of which were highly expressed in cerebellar granule cells (10).To further assess the functions of IP6K2 in the brain, we explored its binding partners using coimmunoprecipitation and tandem liquid chromatography mass spectrometry (LC-MS/MS). Here, we report that IP6K2 robustly interacts with creatine kinase-B (CK-B), which regulates energy homeostasis of cells and exists in two forms, brain type (CK-B) and muscle type (CK-M). CK catalyzes the reversible transfer of the phosphate group of phosphocreatine to ADP to yield ATP (11, 12). A functional interplay between mitochondrial and cytosolic isoforms of CK regulates cellular energy homeostasis. Cytosolic CK rephosphorylates locally produced free ADP and increases creatine globally, while the mitochondrial enzyme catalyzes the conversion of creatine to phosphocreatine utilizing mitochondrial ATP (13–15).Here, we show that IP6K2 loss leads to decreased CK-B expression, reduced ATP levels, and diminished mitochondrial activity associated with increased oxidative stress. About 80 to 90% of ATP is generated in the mitochondria by oxidative phosphorylation, and diminished ATP levels are the immediate effect of mitochondrial dysfunction. Loss of IP6K2 and CK-B reflects the suppression of the mitochondrial cytochrome c1 expression, a component of complex III of the mitochondrial electron transport chain. In the present study, we report a physiologic association of CK-B and IP6K2, whose disruption impacts mitochondrial functions.Dendritic morphogenesis was reduced in IP6K2-deficient neurons and was rescued by restoring normal levels of ATP. These observations reveal an essential role of IP6K2 in the energy production of the brain. Our findings indicate that IP6K2 is a key regulator of mitochondrial homeostasis which promotes neuroprotection.     

Does stress exacerbate liver diseases?

Although anecdotal comments on detrimental effects of psychosocial stress on liver diseases can be found even in the early literature, only recently has scientific evidence been reported. The present article reviewed such evidence to demonstrate how stress exacerbates liver diseases. A search of the literature from the last two decades was performed using MEDLINE by pairing ‘psychological stress’ with ‘liver’ or ‘hepatitis.’ Additional research was conducted by screening the bibliographies of articles retrieved in the MEDLINE search. The search results showed that the principal effectors of the activated hypothalamic–pituitary–adrenal (HPA) axis, glucocorticoids, can exert a facilitative effect on the hepatic inflammatory response and even increase the risk of developing hepatocellular carcinoma. For certain liver diseases, defective HPA axis activation, which probably contributed to the exacerbation of the liver disease, has been reported. The efferent sympathetic/adrenomedullary system mainly contributes to the stress‐induced exacerbation of liver diseases via its neurotransmitters, the catecholamines. In contrast, the efferent parasympathetic nervous system elicits an inhibitory effect on the development of hepatic inflammation. In conclusion, the pathophysiological interaction between stress and the liver appears to be regulated by the complex, dynamic networks of both the endocrine and autonomic nervous systems, which implies a further need for basic research into the involved mechanisms and for clinical evidence to apply psychosocial support to patients with chronic liver diseases.     

Effects of audio stimulation on gastric myoelectrical activity and sympathovagal balance in healthy adolescents and adults

Aim: The primary aim of this study was to investigate the effects of different audio stimulations on gastric myoelectrical activity and sympathovagal balance in adolescents compared with adults. Methods: The study was performed in 11 adults and 12 adolescents. Each subject underwent two sessions, one for classical music, and the other for noise. Each session consisted of 30 min of baseline, 30 min of fasting audio stimulation, a test meal, 30 min of fed audio stimulation, and 30 min of recovery. Electrocardiogram and electrogastrogram were both recorded throughout each session. Results: (i) In the fasting state, both classical music and noise impaired gastric slow wave activity in adolescents. In adults, noise had no effects while classical music moderately improved slow wave rhythmicity. (ii) In the fed state, neither noise nor music had any effects on gastric slow waves. (iii) In the fasting state, both noise and music increased the sympathovagal balance in adolescents; in adults only noise had such an effect. (iv) The test meal increased the sympathovagal balance in all groups. Conclusions: Gastric slow waves and the sympathovagal balance are more strongly affected by audio stimulation in adolescents than in adults. The test meal normalizes the audio stimulation‐induced differences between the groups.     

Relationship between serum levels of oxidative stress and metabolic syndrome components

Aims

This study aimed to investigate the relationship between the total antioxidant capacity (TAC) and Malondialdehyde (MDA) with number of metabolic syndrome (Mets) components on the personnel working in Shahroud University of Medical Sciences.

Presowing seed treatment with melatonin protects red cabbage seedlings against toxic copper ion concentrations

Abstract:  One of the targets of modern plant physiology is to identify tools for improving seed germination and plant growth under unfavorable environmental conditions. Seeds of Brassica oleracea rubrum were pretreated with melatonin at concentrations: 1, 10, and 100 μ m using a hydropriming method. Air-dried seeds of each experimental variants that were nonpretreated (control), hydroprimed (H) or hydroprimed with melatonin (HM1, HM10, and HM100) were germinated in darkness for 3 days at 25°C. Young seedlings were then transferred to the light and grown for an additional 5 days. Both germination and growth tests were performed in water and in CuSO₄ water solutions in concentrations of 0.5 and 1 m m . H, HM1 and HM10 improved seed germination both in water and in the presence of Cu²⁺. One or 10 μ m melatonin eliminated the inhibitory effect of the 0.5 m m metal concentration on the fresh weight of seedlings. HM100 had a negative effect; thus seed germination was lower and seedlings had poor establishment. The toxic effect of Cu²⁺ manifested by membrane peroxidation and DNA endoreplication blocking in the seedlings grown from nontreated (control) and H seeds was not observed in the seedlings grown from HM1 and HM10 seeds; in contrast, HM100 enhanced the toxic effect of Cu²⁺.     

Insulin-sensitizing antihyperglycaemic medications are associated with better outcome in patients with diabetes undergoing cardiac stress testing

Background: There are several treatment modalities available for diabetes; however, the effects of the different medications on coronary artery disease are less understood. The purpose of this study was to evaluate the correlation of insulin‐sensitizing therapy with the outcome of stress myocardial perfusion testing and to compare it with conventional therapy. Methods: Of 417 patients referred to stress testing for evaluation of chest pain, 222 were identified as being treated with conventional therapy only (insulin and insulin secretagogues) and 195 as being treated with insulin sensitizers (metformin and thiazolidinediones (TZD)). Multivariate logistic regression models were used to correct for confounding factors and to determine the independent relation between treatment type and stress‐test outcome. Results: Ischaemia, infarction and the composite outcome were less frequent in the insulin‐sensitizer group than in the conventional therapy group (odds ratio (OR) = 0.39, P = 0.025; OR = 0.32, P = 0.021 and OR = 0.38, P = 0.009, respectively). The subgroup analysis showed that treatment with metformin (n = 125) compared with conventional therapy was associated with less infarction or the composite outcome of ischaemia and/or ischaemia (OR = 0.18 (95% confidence interval (CI): 0.05–0.66), P = 0.010; OR = 0.34 (95%CI: 0.15–0.80), P = 0.014, respectively). Treatment with TZD (n = 43) was associated with a trend to less frequent ischaemia (OR = 0.18 (95%CI: 0.03–1.01), P = 0.051). Conclusion: The addition of insulin‐sensitizing medications to the conventional diabetes therapy or their sole use was associated with decreased coronary artery disease or its severity in patients with diabetes as determined by stress myocardial perfusion study. Randomized prospective trials will be necessary to prove this benefit.     

糖尿病大鼠肾脏组织中氧化应激的实验研究

目的探讨糖尿病大鼠肾脏组织中氧化应激水平在糖尿病肾病中的作用。方法采用STZ诱导糖尿病模型鼠。36只大鼠随机分为正常对照组，糖尿病Ⅰ组(给予鱼精蛋白锌胰岛素2～4U／2d)，糖尿病Ⅱ组(每日给予鱼精蛋白锌胰岛素9～12U／kg体重)。喂养至12w，测定各组血糖，血中总胆固醇(TC)、甘油三酯(TG)、尿素氮、肌酐、糖化血红蛋白(HbAlc)水平，尿肌酐水平和24h尿蛋白含量。运用比色法测定肾脏皮质中抗氧化酶的活性，包括总超氧化物歧化酶(TSOD)、铜锌超氧化物歧化酶(Cu—Zn SOD)、锰超氧化物歧化酶(MnSOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH—Px)，以及丙二醛(MDA)的含量。结果糖尿病Ⅱ组与正常对照组间各指标无显著性差异。糖尿病Ⅰ组与Ⅱ组或正常对照组比较，血糖、TC、TG、HbAlc明显升高；抗氧化酶中，TSOD、Cu—Zn SOD、CAT活性明显下降，GSH—Px活性则升高，Mn SOD活性在各组中无显著性差异；MDA水平明显升高。尿蛋白含量在糖尿病Ⅰ组较其他两组明显升高，而肌酐清除率则明显下降。结论糖尿病可以引起大鼠肾脏氧化应激水平的升高，在糖尿病肾病发病机制中起重要作用。     

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Autophagy is a conserved catabolic process characterized by the de novo generation of a double-membrane structure called an autophagosome with a fundamental function in the bulk turnover of cytoplasmic components, including proteins, RNAs, and organelles. Genetic studies in yeast have elucidated the molecular machinery of autophagy, whereby 42 autophagy-related (ATG) genes have been identified (1–3). These ATG genes are highly conserved among eukaryotes but often have multiple isoforms in other higher organisms, in particular in sessile plants. Albeit increasing understanding on the molecular function of Atg proteins in acting hierarchically on the phagophore assembly site (PAS) to produce autophagosomes, the origin of the autophagosomal membrane remains unclear in higher eukaryotes. Furthermore, the dedication of other membranes and machineries in the autophagy pathway remains under investigation.Plant autophagy is known to play important roles in the sessile lifestyle of plants, participating in seed germination, seedling establishment, plant development, hormone responses, lipid metabolism, and reproductive development (4). Plant autophagy research is advancing with findings not only on the counterparts of the yeast/mammalian Atg proteins but also dealing with some plant-unique factors functioning in different steps of autophagosome biogenesis, thereby uncovering novel mechanisms that might or might not be conserved in nonplant species (5). More interestingly, higher plants possess multiple protein isoforms of ATG machinery, whose functional heterogeneity in the autophagy pathway has only recently been unveiled (6).The coat protein complex II (COPII) machinery consists of five cytosolic components: the small GTPase Sar1, the inner coat protein dimer Sec23-Sec24, and the outer coat proteins Sec13-Sec31. These proteins are essential for COPII-coated vesicle formation, which buds from specialized regions of the ER, namely ER exit sites (ERESs) (7). Under nutrient-rich conditions, COPII vesicles mediate anterograde ER to Golgi transport. However, increasing evidence from yeast and mammals suggests that the COPII machinery or even COPII vesicles themselves may contribute to autophagosome formation when cells are starved for nutrients (8–16). Gene duplication events have occurred substantially in sessile plants during evolution, and the importance of distinct paralogs in environmental stress adaptation during plant development has been implied (17). Arabidopsis encodes multiple COPII paralogs in its genome, including five Sar1s, seven Sec23s, three Sec24s, two Sec13s, and two Sec31s (17). Increasing numbers of studies have pinpointed the functional diversity and importance of distinct COPII paralogs in ER protein export (18–23). Nonetheless, the mechanism by which COPII vesicles are redirected to the autophagy pathway upon nutrient starvation, and their roles in autophagosome biogenesis, remains unclear. Furthermore, the participation of specific COPII paralogs in autophagy regulation remains unknown in plants.Here, we report on a role of a specific Sar1 homolog, AtSar1d, that modulates plant autophagosome biogenesis in concert with AtRabD2a. Large-scale proteomic analysis of the ATG interactome has revealed possible mechanistic connections between the ATG machinery and specific COPII components in plants. Cellular and biochemical analyses have shown that the dominant negative (DN) mutant of AtSar1d (AtSar1dDN) specifically perturbs YFP-ATG8 vacuolar degradation upon autophagic induction. Consistently, a transfer DNA (T-DNA) insertion mutant of AtSar1d exhibited starvation-related phenotypes. Notably, AtSar1d regulates autophagosome progression through specific recognition of ATG8e by a previously uncharacterized noncanonical motif. We further identify a plant-unique Rab1/Ypt1 homolog AtRabD2a that colocalizes with AtSar1d and ATG8 upon starvation by transient expression in Arabidopsis protoplasts. A DN mutant of AtRabD2a (AtRabD2aNI) perturbs autophagy flux, while AtRabD2a is indispensable for bridging the AtSar1d-positive COPII vesicles with the ATG1 complex, thus contributing to autophagosome biogenesis in plants. Our study therefore unequivocally demonstrates that the plant-specific COPII machinery regulates autophagosome biogenesis and sheds light on the evolutionary importance of gene duplication events in the plant autophagy pathway.     

ST-recovery loop of exercise-induced ST deviation in the identification of coronary artery disease: which parameters should we measure?

This study aimed to characterize the ST-recovery loop and assess which range of heart rates (HRs) best discriminates between patients with and without significant coronary artery stenosis. Bicycle exercise tests were undertaken in 44 men and 18 women with coronary artery disease (CAD) and in 59 controls (26 men, 33 women) in the same age range with no signs of CAD. The ST level and the ST-segment slope were continuously monitored, and changes from rest to peak exercise and to 4 min after exercise, respectively, were calculated. Plotting the ST level against HR gives the STHR loop, characterized by the normalized area (NA(alpha)) circumscribed by the ST level during and after exercise from alpha% to 100% of the HR range. Eight values of alpha between 20% and 90% were investigated, and chest and extremity leads were investigated separately. Optimal alpha was found to be < or =70% in men and < or =30% in women. Change in ST-segment slope was the only parameter that gave significant additional discriminating power in both men and women once the area had been taken into account. We conclude that NA(alpha) for extremity and chest leads have similar weights, and that a substantial part of the STHR loop should be taken into consideration, especially in women. NA(30) was superior to end-exercise ST-depression and STHR loop orientation (as defined by the sign of NA(90)) in both men and women, and to ST/HR index in men, in identifying CAD.