Free fatty acids (FFAs) are important substrates for mitochondrial oxidative metabolism and ATP synthesis but also cause serious stress to various tissues, contributing to the development of metabolic diseases. CD36 is a major mediator of cellular FFA uptake. Inside the cell, saturated FFAs are able to induce the production of cytosolic and mitochondrial reactive oxygen species (ROS), which can be prevented by co-exposure to unsaturated FFAs. There are close connections between oxidative stress and organellar Ca2+ homeostasis. Highly oxidative conditions induced by palmitate trigger aberrant endoplasmic reticulum (ER) Ca2+ release and thereby deplete ER Ca2+ stores. The resulting ER Ca2+ deficiency impairs chaperones of the protein folding machinery, leading to the accumulation of misfolded proteins. This ER stress may further aggravate oxidative stress by augmenting ER ROS production. Secondary to ER Ca2+ release, cytosolic and mitochondrial matrix Ca2+ concentrations can also be altered. In addition, plasmalemmal ion channels operated by ER Ca2+ depletion mediate persistent Ca2+ influx, further impairing cytosolic and mitochondrial Ca2+ homeostasis. Mitochondrial Ca2+ overload causes superoxide production and functional impairment, culminating in apoptosis. This vicious cycle of lipotoxicity occurs in multiple tissues, resulting in β-cell failure and insulin resistance in target tissues, and further aggravates diabetic complications.

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨm hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.

Background: Mitochondrial Ca 2ϩ uptake affects energy metabolism and insulin secretion. Results: Knockdown of mitochondrial Ca 2ϩ uniporter decreases respiratory chain activity and mitochondrial pH gradient generation. Conclusion: Mitochondrial Ca 2ϩ uptake via uniporter is essential for oxidative phosphorylation and metabolism-secretion coupling. Significance: The present study identifies mechanisms of action and bioenergetic consequences of mitochondrial Ca 2ϩ transporters in insulin-releasing cells.

TGF-␤ is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-␤1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-␤1-induced apoptosis in immortalized podocytes. TGF-␤1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-␤ receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTOR⌬ augmented TGF-␤1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-␤1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-␤1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-␤1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-␤1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-␤1-induced ERK1/2-mTOR activation. Our data suggest that TGF-␤1 increases translation ofNox4throughtheSmad-ERK1/2-mTORC1axis,whichisindependent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic kidney diseases.Podocyte damage is one of the key determinants of the majority of diabetic and non-diabetic glomerular diseases, leading to chronic kidney diseases and end-stage renal disease (1). Leakage of plasma proteins in the urine indicates the onset of renal diseases that are associated with podocyte injury (2-4). Recent evidence has shown that mammalian target of rapamycin (mTOR) 3 signaling activation is an important mediator of diabetic nephropathy in mice and humans (5, 6). Increased mTOR activity has been reported in different glomerular diseases, and mTORC1 inhibition by rapamycin and everolimus shows beneficial effects against diabetic nephropathy, focal segmental glomerulosclerosis, minimal change disease, membranous nephropathy, etc. (7-10). But contrasting evidence also indicates that mTORC1 inhibition by the immunosuppressive drug rapamycin leads proteinuria and podocyte apoptosis and develops primary focal segmental glomerulosclerosis in renal transplant recipients (11). Therefore, it might be important to investigate the detailed molecular mechanisms to...

Background and ObjectivesExperimental protocols for remote ischemic conditioning (RIC) utilize models in which a tourniquet is placed around the hindlimb or effluent is collected from an isolated heart. In analyzing the humoral factors that act as signal transducers in these models, sampled blood can be influenced by systemic responses, while the effluent from an isolated heart might differ from that of the hindlimb. Thus, we designed a new isolated hindlimb model for RIC and tested whether the effluent from this model could affect ischemia/reperfusion (IR) injury and if the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways are involved in RIC.Materials and MethodsAfter positioning needles into the right iliac artery and vein of rats, Krebs-Henseleit buffer was perfused using a Langendorff apparatus, and effluent was collected. The RIC protocol consisted of 3 cycles of IR for 5 minutes. In the RIC effluent group, collected effluent was perfused in an isolated heart for 10 minutes before initiating IR injury.ResultsCompared with the control group, the infarct area in the RIC effluent group was significantly smaller (31.2%±3.8% vs. 20.6%±1.8%, p<0.050), while phosphorylation of signal transducer and activation of transcription-3 (STAT-3) was significantly increased. However, there was a trend of increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in this group.ConclusionThis is the first study to investigate the effect of effluent from a new isolated hindlimb model after RIC on IR injury in an isolated heart model. The RIC effluent was effective in reducing the IR injury, and the cardioprotective effect was associated with activation of the SAFE pathway.