Background-The modification of proteins with O-linked ␤-N-acetylglucosamine (O-GlcNAc

Abstract-Uncoupling proteins (UCPs) are located in the mitochondrial inner membrane and partially dissipate the transmembrane proton electrochemical gradient. UCP2 is expressed in various human and rodent tissues, including the heart, where its functional role is unknown. In the present study, we tested the hypothesis that UCP2 overexpression could protect cardiomyocytes from oxidative stress-induced cell death by reducing reactive oxygen species (ROS) production in mitochondria. Using an adenoviral vector containing human UCP2, we investigated the effects of UCP2 overexpression on the mitochondrial death pathway induced by oxidative stress (100 mol/L H 2 O 2 ) in cultured neonatal cardiomyocytes. UCP2 overexpression significantly suppressed markers of cell death, including TUNEL positivity, phosphatidylserine exposure, propidium iodide uptake, and caspase-3 cleavage. Furthermore, UCP2 remarkably prevented the catastrophic loss of mitochondrial inner membrane potential induced by H 2 O 2 , which is a critical early event in cell death. Ca 2ϩ overload and the production of ROS in mitochondria, both of which contribute to mitochondrial inner membrane potential loss, were dramatically attenuated by UCP2 overexpression. Thus, overexpression of UCP2 attenuates ROS generation and prevents mitochondrial Ca 2ϩ overload, revealing a novel mechanism of cardioprotection.

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Abstract-Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. In the present study, we characterize three distinct phases of the H 2 O 2 -induced response, which leads to loss of mitochondrial membrane potential (⌬⌿ m ) and subsequent cell death in cultured cardiac myocytes.(1) Priming: After H 2 O 2 exposure (100 mol/L), cells maintain a constant ⌬⌿ m for the cell-to-cell specific latency but at the same time undergo progressive changes in inner mitochondrial membrane structure (swelling and loss of cristae by electron microscopy). An increase of matrix calcium is required, but not sufficient, for this process. (2) Depolarization: Priming is followed by sudden depolarization of ⌬⌿ m , which is mediated by mitochondrial permeability transition pore opening, as evidenced by the concomitant release of calcein from mitochondria. This process is rapid (Ͻ4 minutes), complete, and irreversible. The duration of depolarization is constant and does not depend on the length of the priming process in any given cell.

We examined the effects of chronic centrally administered leptin on the glucose metabolism of streptozotocin-induced diabetic (STZ-D) rats, a model for insulin-dependent diabetes mellitus. When 3 microg.rat(-1).day(-1) of leptin was infused into the third ventricle for 6 consecutive days (STZ-LEP), STZ-D rats became completely euglycemic. The effect was not seen when the same dosage was administered s.c. Centrally administered leptin did not affect peripheral insulin levels. The feeding volume of STZ-LEP rats was suppressed to the level of non-STZ-D control rats. No improvement of hyperglycemia was noted when STZ-D rats were pair-fed to match the feeding volume of STZ-LEP rats. Thus, the euglycemia of STZ-LEP rats cannot be due to the decreased feeding volume. In the STZ-D rat, glucokinase mRNA, a marker of glycolysis, is down-regulated whereas glucose-6-phosphatase mRNA, a marker of gluconeogenesis, and glucose transporter (GLUT) 2, which is implicated in the release of glucose from liver, are up-regulated. GLUT4, uncoupling protein (UCP) 1, and UCP3 were down-regulated in brown adipose tissue. These parameters returned to normal upon central infusion of leptin. GLUT4 was not down-regulated in the skeletal muscle of STZ-D rats; however, fatty acid binding protein and carnitine palmitoyltransferase I, markers for utilization and beta-oxidation of fatty acids, were up-regulated and restored when the rats were treated with leptin. The increase and subsequent decrease of fatty acid utilization suggests a decrease of glucose uptake in the skeletal muscle of STZ-D rats, which was restored upon central leptin administration. We conclude that centrally infused leptin does not control serum glucose by regulating feeding volume or elevating peripheral insulin, but by regulating hepatic glucose production, peripheral glucose uptake, and energy expenditure. The present study indicates the possibility of future development of a new class of anti-diabetic agents that act centrally and independent of insulin action.

Background— The Na + -H + exchanger figures prominently in cardiac ischemia-reperfusion injury. Several experimental and clinical studies have demonstrated a cardioprotective effect of Na + -H + exchanger inhibition; however, the precise mechanisms have not been established. Methods and Results— We examined the effects of cariporide (HOE642, 10 μmol/L) on cell death induced by oxidative stress (H 2 O 2 , 100 μmol/L) in cultured neonatal rat cardiomyocytes. Cariporide significantly suppressed markers of cell death, such as TUNEL positivity and caspase-3 cleavage, at 8 or 16 hours after H 2 O 2 . The early phase of cell death, reported by increases in phosphatidylserine exposure and propidium iodide uptake, was also inhibited by cariporide. To explore the mechanisms of cell protection, we examined the effects of cariporide on increases in intracellular Na + and Ca 2+ induced by oxidative stress. Cariporide remarkably suppressed cytosolic Na + and Ca 2+ accumulation. Next, we investigated the effects of cariporide on mitochondria-associated death process. Mitochondrial Ca 2+ overload induced by H 2 O 2 was remarkably suppressed by cariporide. Loss of mitochondrial membrane potential is a critical step of the death pathway; cariporide prevented mitochondrial membrane potential loss induced by H 2 O 2 . Conclusions— Cariporide protects cardiomyocytes against oxidant-induced cell death by preserving intracellular ion homeostasis and mitochondrial integrity.

Background and Purpose-Mitochondrial ATP-sensitive potassium (mitoK ATP ) channels are present in the brain, and several reports have shown that mitoK ATP channel openers protect the brain against ischemic injury. However, the precise mechanisms of this protection are not well established. We hypothesized that mitoK ATP channel openers prevent apoptosis by preserving mitochondrial membrane potential. Methods-We investigated the effect of mitoK ATP channel openers on apoptosis induced by oxidative stress using cultured cerebellar granule neurons. Results-The mitoK ATP channel opener diazoxide (100 mol/L) significantly suppressed the number of cells with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive nuclei and the increase in caspase-3 activity induced by 20 mol/L H 2 O 2 . Diazoxide and another opener, pinacidil, prevented the loss of mitochondrial inner membrane potential (⌬⌿ m ) induced by H 2 O 2 . These effects were abolished by 5-hydroxydecanoate (500 mol/L), a mitoK ATP channel blocker. Cyclosporin A and bongkrekic acid, inhibitors of the mitochondrial permeability transition pore, also prevented ⌬⌿ m loss, confirming the involvement of the mitochondrial permeability transition in the apoptotic cascade in neurons. Furthermore, diazoxide prevented the increase in extracellular glutamate concentration induced by H 2 O 2 , but this effect was not attributable to activation of surface K ATP channels. Conclusions-MitoK ATP channel openers inhibited apoptosis by preserving mitochondrial inner membrane potential. These beneficial effects may suggest a possible new target for neuroprotection.

Glucose fluctuations increase the incidence of AF by promoting cardiac fibrosis. Increased ROS levels caused by upregulation of Txnip expression may be a mechanism whereby in glucose fluctuations induce fibrosis.