Kyu-Sang Park

Influence of OATP1B1 Genotype on the Pharmacokinetics of Rosuvastatin in Koreans

This study was carried out to determine whether polymorphisms of organic anion‐transporting polypeptide 1B1 (OATP1B1) have an effect on rosuvastatin pharmacokinetics in Koreans. Among 200 subjects genotyped for OATP1B1 c.388A>G, and c.521T>C, 30 subjects were selected for the rosuvastatin pharmacokinetic study. The area under the concentration–time curve for 0 to infinity (AUC0–∞) of rosuvastatin for group 1 (*1a/*1a, *1a/*1b, *1b/*1b), group 2 (*1a/*15, *1b/*15), and group 3 (*15/*15) were 111±49.3, 126±45.2, and 191±31.0 ng h/ml, respectively, with significant differences among the three groups (P=0.0429) and between *15/*15 and the other groups (P=0.0181). The maximum plasma concentration (Cmax) also showed a significant difference between *15/*15 and the other groups (P=0.0181). There were no significant differences in rosuvastatin‐lactone pharmacokinetics among the three groups. The pharmacokinetic exposure of rosuvastatin was higher in the OATP1B1*15/*15 subjects than the others, suggesting a potential association between the OATP1B1 genetic polymorphisms and altered rosuvastatin pharmacokinetics in Korean populations.

Selective actions of mitochondrial fission/fusion genes on metabolism-secretion coupling in insulin releasing cells

Mitochondria form filamentous networks that undergo continuous fission/fusion. In the pancreatic β-cells, mitochondria are essential for the transduction of signals linking nutrient metabolism to insulin granule exocytosis. Here we have studied mitochondrial networks in the insulinoma cell line INS-1E, primary rat and human β-cells. We have further investigated the impact of mitochondrial fission/fusion on metabolism-secretion coupling in INS-1E cells. Overexpression of hFis1 caused dramatic mitochondrial fragmentation, whereas Mfn1 evoked hyperfusion and the aggregation of mitochondria. Cells overexpressing hFis1 or Mfn1 showed reduced mitochondrial volume, lowered cellular ATP levels, and as a consequence, impaired glucose-stimulated insulin secretion. Decreased mitochondrial ATP generation was partially compensated for by enhanced glycolysis as indicated by increased lactate production in these cells. Dominant-negative Mfn1 elicited mitochondrial shortening and fragmentation of INS-1E cell mitochondria, similar to hFis1. However, the mitochondrial volume, cytosolic ATP levels, and glucose-stimulated insulin secretion were little affected. We conclude that mitochondrial fragmentation per se does not impair metabolism-secretion coupling. Through their impact on mitochondrial bioenergetics and distribution, hFis1 and Mfn1 activities influence mitochondrial signal generation thereby insulin exocytosis.

FCCP depolarizes plasma membrane potential by activating proton and Na + currents in bovine aortic endothelial cells

We investigated the effects of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore and uncoupler of mitochondrial oxidative phosphorylation in mitochondria, on plasma membrane potential and ionic currents in bovine aortic endothelial cells (BAECs). The membrane potential and ionic currents of BAECs were recorded using the patch-clamp technique in current-clamp and voltage-clamp modes, respectively. FCCP activated ionic currents and depolarized the plasma membrane potential in a dose-dependent manner. Neither the removal of extracellular Ca2+ nor pretreatment with BAPTA/AM affected the FCCP-induced currents, implying that the currents are not associated with the FCCP-induced intracellular [Ca2+]i increase. FCCP-induced currents were significantly influenced by the changes in extracellular or intracellular pH; the increased proton gradient produced by lowering the extracellular pH or intracellular alkalinization augmented the changes in membrane potential and ionic currents caused by FCCP. FCCP-induced currents were significantly reduced under extracellular Na+-free conditions. The reversal potentials of FCCP-induced currents under Na+-free conditions were well fitted to the calculated equilibrium potential for protons. Interestingly, FCCP-induced Na+ transport (subtracted currents, Icontrol–INa+-free) was closely dependent on extracellular pH, whereas FCCP-induced H+ transport was not significantly affected by the absence of Na+. These results suggest that the FCCP-induced ionic currents and depolarization, which are strongly dependent on the plasmalemmal proton gradient, are likely to be mediated by both H+ and Na+ currents across the plasma membrane. The relationship between H+ and Na+ transport still needs to be determined.

Rifampin Enhances the Glucose‐Lowering Effect of Metformin and Increases OCT1 mRNA Levels in Healthy Participants

We evaluated the effect of the pregnane X receptor (PXR) agonist rifampin on metformin pharmacokinetics, organic cation transporter 1 (OCT1) and OCT2 mRNA levels, and glucose levels, using the oral glucose tolerance test (OGTT) in 16 healthy subjects. The glucose‐lowering effects of metformin were evaluated by OGTT before and after metformin treatment on days 1 and 2 and again on days 13 and 14 after a 10‐day course of rifampin. Rifampin increased the difference in maximum glucose levels (ΔGmax) by 41.9% (P = 0.024) and the area under the concentration–time curve (AUC) during the first 60 min after glucose ingestion (ΔAUCgluc60) by 54.5% (P = 0.020). Renal clearance (CLR) of metformin was increased by 16% (P = 0.008), but the systemic exposure was only slightly increased (13%, P = 0.049), possibly because of increased absorption. Rifampin increased OCT1 mRNA levels 4.1‐fold in peripheral blood cells (P = 0.001); however, OCT2 mRNA was not detected. Our results suggest that rifampin increases OCT1 expression and hepatic uptake of metformin, leading to enhanced glucose‐lowering action.

Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal beta cells

Glucose‐evoked mitochondrial signals augment ATP synthesis in the pancreatic β cell. This activation of energy metabolism increases the cytosolic ATP/ADP ratio, which stimulates plasma membrane electrical activity and insulin granule exocytosis. We have recently demonstrated that matrix pH increases during nutrient stimulation of the pancreatic β cell. Here, we have tested whether mitochondrial matrix pH controls oxidative phosphorylation and metabolism‐secretion coupling in the rat β‐cell line INS‐1E. Acidification of the mitochondrial matrix pH by nigericin blunted nutrient‐dependent respiratory and ATP responses (continuously monitored in intact cells). Using electrophysiology and single cell imaging, we find that the associated defects in energy metabolism suppress glucose‐stimulated plasma membrane electrical activity and cytosolic calcium transients. The same parameters were unaffected after direct stimulation of electrical activity with tolbutamide, which bypasses mitochondrial function. Furthermore, lowered matrix pH strongly inhibited sustained, but not first‐phase, insulin secretion. Our results demonstrate that the matrix pH exerts a control function on oxidative phosphorylation in intact cells and that this mode of regulation is of physiological relevance for the generation of downstream signals leading to insulin granule exocytosis. We propose that matrix pH serves a novel signaling role in sustained cell activation.—Akhmedov, D., Braun, M., Mataki, C., Park, K.‐S., Pozzan, T., Schoonjans, K., Rorsman, P., Wollheim, C. B., Wiederkehr, A. Mitochondrial matrix pH controls oxidative phosphorylation and metabolism‐secretion coupling in INS‐1E clonal β cells. FASEB J. 24, 4613–4626 (2010). www.fasebj.org

Upregulation of mitochondrial Nox4 mediates TGF-β-induced apoptosis in cultured mouse podocytes.

Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β1 markedly depolarized mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. These results suggest that TGF-β1-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.

Soluble α-klotho as a novel biomarker in the early stage of nephropathy in patients with type 2 diabetes.

Objective Although α-klotho is known as an anti-aging, antioxidant, and cardio-renal protective protein, the clinical implications of soluble α-klotho levels in patients with diabetes have not been evaluated. Therefore, this study evaluated whether plasma and urinary α-klotho levels are associated with albuminuria in kidney disease in diabetes. Research Design and Methods A total of 147 patients with type 2 diabetes and 25 healthy control subjects were enrolled. The plasma and urine concentrations of α-klotho were analyzed by enzyme-linked immunosorbent assay. Results Plasma α-klotho (572.4 pg/mL [95% CI, 541.9–604.6 pg/mL] vs. 476.9 pg/mL [95% CI, 416.9–545.5 pg/mL]) and urinary α-klotho levels (59.8 pg/mg creatinine [95% CI, 43.6–82.0 pg/mg creatinine] vs. 21.0 pg/mg creatinine [95% CI, 9.7–45.6 pg/mg creatinine]) were significantly higher in diabetic patients than non-diabetic controls. Among diabetic patients, plasma α-klotho concentration was inversely associated with albuminuria stages (normoalbuminuria, 612.6 pg/mL [95% CI, 568.9–659.6 pg/mL], microalbuminuria, 551.8 pg/mL [95% CI, 500.5–608.3 pg/mL], and macroalbuminuria, 505.7 pg/mL [95% CI, 439.7–581.7 pg/mL] (p for trend  = 0.0081), while urinary α-klotho levels were remained constantly high with increasing urinary albumin excretion. Conclusions Soluble α-klotho levels in plasma and urine may be novel and useful early markers of diabetic renal injury.

Essential Role of Mitochondrial Ca2+ Uniporter in the Generation of Mitochondrial pH Gradient and Metabolism-Secretion Coupling in Insulin-releasing Cells

Background: Mitochondrial Ca2+ uptake affects energy metabolism and insulin secretion. Results: Knockdown of mitochondrial Ca2+ uniporter decreases respiratory chain activity and mitochondrial pH gradient generation. Conclusion: Mitochondrial Ca2+ 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 Ca2+ transporters in insulin-releasing cells. In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca2+ influx, which triggers insulin exocytosis. The mitochondrial Ca2+ uniporter (MCU) mediates Ca2+ uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca2+ rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca2+. Suppression of the putative Ca2+/H+ antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca2+-induced matrix acidification. These results demonstrate that MCU-mediated Ca2+ uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.

Biological Role of Anti-aging Protein Klotho.

Klotho-deficient mice have accelerated aging phenotypes, whereas overexpression of Klotho in mice extends lifespan. Klotho is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble Klotho decrease with age, and the klotho gene is associated with increased risk of age-related diseases. The three forms of Klotho protein have distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors, functions as an obligatory co-receptor for FGF23, which is involved in aging and the development of chronic diseases via regulation of Pi and vitamin D metabolism. Secreted Klotho functions as a humoral factor with pleiotropic activities including regulation of oxidative stress, growth factor signaling, and ion homeostasis. Secreted Klotho is also involved in organ protection. The intracellular form of Klotho suppresses inflammation-mediated cellular senescence and mineral metabolism. Herein we provide a brief overview of the structure and function and recent research about Klotho.

Amiloride derivatives induce apoptosis by depleting ER Ca2+ stores in vascular endothelial cells

Background and purpose:  Amiloride derivatives are blockers of the Na+/H+ exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action.