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Hyperglycemia Primes Cells for Programmed Cell Death Shift in a Glycolysis-Dependent Manner
429 Hyperglycemia Primes Cells for Programmed Cell Death Shift in a Glycolysis-Dependent Manner Nicole L Shakerley1, Tori Smiraglia1, Katherine Walker1, Miranda Craft1, and Timothy J LaRocca1 1 Albany College of Pharmacy and Health Sciences, USA Necroptosis is a RIP1-dependent programmed cell death (PCD) pathway distinct from apoptosis. Downstream effector pathways of necroptosis include formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), both of which are influenced by glycolysis, suggesting that increased cellular glucose may prime necroptosis. Here, we establish that exposure to hyperglycemic levels of glucose enhances necroptosis in U937 monocytes. Pharmacologic or siRNA inhibition of RIP1 prevented the enhanced death, confirming it as RIP1-dependent necroptosis. Hyperglycemia induces a shift from apoptotic death to necroptosis in response to the extrinsic apoptotic stimulus TNFα. Increased membrane permeability and decreased phosphatidylserine exposure were seen in the hyperglycemic condition as compared to euglycemia. Additionally, cell death was shown to be caspase dependent under euglycemic conditions and shifted to RIP1 dependency in hyperglycemic conditions. Total levels of RIP1, RIP3, and MLKL increased following treatment with high levels of glucose suggesting a potential positive feedback mechanism. Hyperglycemic enhancement of necroptosis depends upon glycolysis with AGEs and ROS playing a role. Furthermore, the hyperglycemic state resulted in dysregulated calcium flux in a ROS dependent manner. Hyperglycemia resulted in increased infarct size in a mouse model of brain hypoxia-ischemia injury. The increased infarct size was prevented by inhibition of RIP1 with nec1s, strongly suggesting that increased necroptosis accounts for exacerbation of this injury in conditions of hyperglycemia. This work reveals that hyperglycemia represents a condition in which cells are extraordinarily susceptible to necroptosis, that local glucose levels alter the balance of PCD pathways, and that clinically relevant outcomes may depend on glucose-mediated effects on PCD.
doi: 10.1016/j.freeradbiomed.2016.10.470 430 Reactive Oxygen Species-Mediated Glucose Metabolic Reprogram Induces Insulin Resistance in Type 2 Diabetes Meiling Wu1 and Dongyun Shi1 1 Shanghai Medical College of Fudan University, People's Republic of China Oxidative stress is known to contribute to insulin resistance in diabetes, however the mechanism is not clear. Here we show that reactive oxygen species (ROS) could reprogram the glucose metabolism through upregulating the pentose pathway so as to induce insulin resistance in type 2 diabetes (T2DM). By using streptozotocin-high fat diet (STZ-HFD) induced T2DM in rats, we show that diabetic rats exhibited high level of oxidative stress accompanied with insulin resistance. Hypoxia inducible factor (HIF1 a ) protein expression as well as its downstream target glucokinase (GK), were upregulated; The glycogen synthesis increased accordingly; However the glycolysis was inhibited as indicated by decreased phosphofructokinase-1 (PFK-1), pyruvate
S178
kinase (PK), phospho-PFK-2/PFK-2 (p-PFK-2/PFK-2) ratio, lactate dehydrogenase (LDH) and pyruvate dehydrogenase kinase (PDK); Pyruvate dehydrogenase (PDH) which promotes pyruvate to generate acetyl-CoA declined as well. While phospho-acetyl-CoA carbox-ylase/acetyl-CoA carboxylase (p-ACC/ACC) ratio increased, meaning that lipid beta-oxidation increased. The pentose pathway was activated as indicated by increased G6PD activity and NADPH level. Our results suggest that diabetic rats countervail ROS stress through increasing pentose pathway, and reprogram the energy metabolic pathway from glycolysis into lipid oxidation in order to compensate the ATP requirement of the body, which causes insulin resistance.
doi: 10.1016/j.freeradbiomed.2016.10.471 431 Enhanced Antioxidant-Defense Preserves Cardiac Dysfunction via Regulation of Cytosolic Levels of Zn and Ca Ions in Hyperglycemic Cardiomyocytes Belma Turan1 and Erkan Tuncay1 Ankara University Faculty of Medicine, Turkey
1
It is well accepted that chronic hyperglycemia is an important risk factor for myocardial infarction and importantly, both acute and chronic hyperglycemia trigger several biochemical and electrophysiological changes resulting an impaired cardiac contractile function. Hypeglycemia first initiates repeated acute changes in cellular metabolism, and then followed by cumulative long-term changes in macromolecules. The long-term changes include mainly a big amount of increases in the production of reactive oxygen species, ROS, which then induce a diabetic tissue/cell damage in several target organs including heart. Marked changes in cytosolic free Zn2+ occurs during contractile activity and Zn2+ movements are controlled by changes in cytosolic free Ca2+, being highly sensitive to cellular oxidative-state in cardiomyocytes. In here, we examined whether cellular antioxidant-defence enhancement preserves cardiac dyfunction via regulation of both diastolic free Zn2+ and Ca2+. The N-acetyl cysteine (NAC)-treatment of diabetic rats led to a balanced oxidant/antioxidant level in both heart and circulation and prevented the altered cellular redox-state. It also prevented diabetes-induced tissue damage and markedly increased diastolic function with marked normalizations in the resting levels of both diastolic free Zn2+ and Ca2+. Both in vivo and in vitro NAC-treatment of diabetic samples prevented the altered kinetic parameters of transient changes in both free Zn 2+and Ca2+ under electrical stimulation, as well as spatio-temporal properties of both local changes of free Zn2+and Ca2+ (sparks) in resting cells. Biochemical analysis demonstrated that NAC-treatment of diabetic rats also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10µM ZnCl2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentrationdependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB can be activated if the cells are exposed directly to Zn2+. We thus for the first time report that an enhancement of antioxidant-defence in diabetics via directly targeting heart, seems to prevent diastolic dysfunction, associated with normalization of RyR2 macromolecular-complex, and thereby prevention of both Zn2+ and Ca2+ leaks via leading to normalization of both diastolic free Zn2+ and Ca2+in cardiomyocytes (Supported with Tübitak SBAG-214S254.
SfRBM / SFRRI 2016
doi: 10.1016/j.freeradbiomed.2016.10.470 430 Reactive Oxygen Species-Mediated Glucose Metabolic Reprogram Induces Insulin Resistance in Type 2 Diabetes Meiling Wu1 and Dongyun Shi1 1 Shanghai Medical College of Fudan University, People's Republic of China Oxidative stress is known to contribute to insulin resistance in diabetes, however the mechanism is not clear. Here we show that reactive oxygen species (ROS) could reprogram the glucose metabolism through upregulating the pentose pathway so as to induce insulin resistance in type 2 diabetes (T2DM). By using streptozotocin-high fat diet (STZ-HFD) induced T2DM in rats, we show that diabetic rats exhibited high level of oxidative stress accompanied with insulin resistance. Hypoxia inducible factor (HIF1 a ) protein expression as well as its downstream target glucokinase (GK), were upregulated; The glycogen synthesis increased accordingly; However the glycolysis was inhibited as indicated by decreased phosphofructokinase-1 (PFK-1), pyruvate
S178
kinase (PK), phospho-PFK-2/PFK-2 (p-PFK-2/PFK-2) ratio, lactate dehydrogenase (LDH) and pyruvate dehydrogenase kinase (PDK); Pyruvate dehydrogenase (PDH) which promotes pyruvate to generate acetyl-CoA declined as well. While phospho-acetyl-CoA carbox-ylase/acetyl-CoA carboxylase (p-ACC/ACC) ratio increased, meaning that lipid beta-oxidation increased. The pentose pathway was activated as indicated by increased G6PD activity and NADPH level. Our results suggest that diabetic rats countervail ROS stress through increasing pentose pathway, and reprogram the energy metabolic pathway from glycolysis into lipid oxidation in order to compensate the ATP requirement of the body, which causes insulin resistance.
doi: 10.1016/j.freeradbiomed.2016.10.471 431 Enhanced Antioxidant-Defense Preserves Cardiac Dysfunction via Regulation of Cytosolic Levels of Zn and Ca Ions in Hyperglycemic Cardiomyocytes Belma Turan1 and Erkan Tuncay1 Ankara University Faculty of Medicine, Turkey
1
It is well accepted that chronic hyperglycemia is an important risk factor for myocardial infarction and importantly, both acute and chronic hyperglycemia trigger several biochemical and electrophysiological changes resulting an impaired cardiac contractile function. Hypeglycemia first initiates repeated acute changes in cellular metabolism, and then followed by cumulative long-term changes in macromolecules. The long-term changes include mainly a big amount of increases in the production of reactive oxygen species, ROS, which then induce a diabetic tissue/cell damage in several target organs including heart. Marked changes in cytosolic free Zn2+ occurs during contractile activity and Zn2+ movements are controlled by changes in cytosolic free Ca2+, being highly sensitive to cellular oxidative-state in cardiomyocytes. In here, we examined whether cellular antioxidant-defence enhancement preserves cardiac dyfunction via regulation of both diastolic free Zn2+ and Ca2+. The N-acetyl cysteine (NAC)-treatment of diabetic rats led to a balanced oxidant/antioxidant level in both heart and circulation and prevented the altered cellular redox-state. It also prevented diabetes-induced tissue damage and markedly increased diastolic function with marked normalizations in the resting levels of both diastolic free Zn2+ and Ca2+. Both in vivo and in vitro NAC-treatment of diabetic samples prevented the altered kinetic parameters of transient changes in both free Zn 2+and Ca2+ under electrical stimulation, as well as spatio-temporal properties of both local changes of free Zn2+and Ca2+ (sparks) in resting cells. Biochemical analysis demonstrated that NAC-treatment of diabetic rats also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10µM ZnCl2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentrationdependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB can be activated if the cells are exposed directly to Zn2+. We thus for the first time report that an enhancement of antioxidant-defence in diabetics via directly targeting heart, seems to prevent diastolic dysfunction, associated with normalization of RyR2 macromolecular-complex, and thereby prevention of both Zn2+ and Ca2+ leaks via leading to normalization of both diastolic free Zn2+ and Ca2+in cardiomyocytes (Supported with Tübitak SBAG-214S254.
SfRBM / SFRRI 2016