Oleic Acid Activates Mitochondrial Energy Metabolism and Reduces Oxidative Stress Resistance in the Pancreatic ß-Cell Line INS-1.

Elevated concentration of saturated fatty acids in plasma adversely affects pancreatic ß-cells, but the effects of unsaturated fatty acids are controversial. In this study, we examined the effects of oleic acid (OA), a monounsaturated fatty acid, on mitochondrial function, which is important for insulin secretion, using INS-1 cells, a pancreatic ß-cell line derived from rats. Observations of mitochondrial membrane potential and intracellular ATP concentration showed that the electron transport chain was enhanced and ATP production increased in cells treated with OA, indicating that the response that occurs from sensing an increase in glucose concentration to the production of ATP was accelerated. Measurements of intracellular reactive oxygen species (ROS) indicated that the rate of increase in ROS after glucose stimulation was significantly higher in OA-treated cells. The mRNA expression levels of superoxide dismutase 1 and 2, which are responsive to ROS and other substances, were significantly increased in OA 1-d treated cells, but decreased in OA 7-d treated cells. It can be inferred that continued exposure to high concentrations of OA reduced ROS processing capacity and increased intracellular ROS levels. The mRNA expression of apoptosis-inducing enzyme Caspase-3 was significantly increased in OA-treated cells, although its activity was not high. However, the apoptosis induction rate after H2O2 stimulation was significantly higher in OA-treated cells. The high OA environment was shown to promote mitochondrial energy metabolism, leading to an increase in glucose sensitivity and a decrease in oxidative stress resistance.

Barley consumption under a high-fat diet suppresses lipogenic genes through altered intestinal bile acid composition.

We evaluated whether barley flour consumption in a high-fat environment affects lipid metabolism through signals mediated by bile acids. Four-week-old mice were fed a high-fat diet supplemented with cellulose (HC) or ß-glucan-rich barley flour (HB) for 12 weeks. Bile acid composition in the intestinal tract and feces was measured by GC/MS. Gene expression levels involved in bile acid metabolism in the liver and intestinal tract were determined by RT-PCR. Similar parameters were measured in mice treated with antibiotics (antibiotics-cellulose [AC] and antibiotics-barley [AB]) to reduce the activity of intestinal bacteria. The Results showed that the HB group had lower liver blood cholesterol and triglyceride levels than the HC group. The HB group showed a significant decrease in primary bile acids in the gastrointestinal tract compared to the HC group. On the other hand, the concentration of secondary bile acids relatively increased in the cecum and feces. In the liver, Fxr activation suppressed gene expression levels in synthesizing bile acids and lipids. Furthermore, in the gastrointestinal tract, Tgr5 was activated by increased secondary bile acids. Correspondingly, AMP levels were increased in the HB group compared to the HC group, AMPK was phosphorylated in the liver, and gene expression involved in lipid synthesis was downregulated. A comparison of the AC and AB groups treated with antibiotics did not confirm these effects of barley intake. In summary, our results suggest that the prevention of lipid accumulation by barley consumption involves signaling through changes in bile acid composition in the intestinal tract.

Consumption of barley flour increases gut fermentation and improves glucose intolerance via the short-chain fatty acid receptor GPR43 in obese male mice.

Barley consumption is expected to increase insulin sensitivity by increasing the level of short-chain fatty acids (SCFAs) and promoting the secretion of GLP-1. However, the involvement of GPR43, a receptor for SCFAs, has not been investigated. Therefore, we evaluated whether the inhibitory effect of ß-glucan-rich barley intake on blood glucose rise is mediated by GPR43 signalling via an increase of SCFAs. C57BL/6J mice and GPR43-knockout mice were fed high-fat diets with either cellulose (HC) or ß-glucan-rich barley flour (HB) for 12 weeks. The level of SCFAs in cecum contents was measured and the concentration of GLP-1 in the portal vein was determined. The supernatant of the cecum contents of C57BL/6J mice was added to GLUTag cells, and then the changes to GLP-1 and intracellular Ca2+ concentrations determined. The same parameters were measured using cells in which GPR43 was knocked down by siRNA. C57BL/6J mice fed HB diets showed a suppressed glucose rise compared to those on the HC diet. Cecum SCFAs and GLP-1 concentration in the portal vein were also increased by the HB diet. When an aqueous solution from the cecum content of mice fed a HB diet was added to GLUTag cells, GLP-1 secretion and intracellular Ca2+ concentration were increased. These phenomena were not observed in cells with knockdown of GPR43. In GPR43 knockout mice an increase of GLP-1 in the portal vein and suppression of blood glucose elevation was attenuated, despite increased SCFAs brought on by the HB diet. In conclusion, GPR43 activation in the intestinal tract via increased SCFAs is required for the glucose intolerance-improving effect of barley consumption.

Fluorescence Analysis of the Mitochondrial Effect of a Plasmalemmal Na+/Ca2+ Exchanger Inhibitor, SEA0400, in Permeabilized H9c2 Cardiomyocytes.

We investigated the effect on mitochondrial Ca2+ of SEA0400, an inhibitor of the Na+/Ca2+ exchanger (NCX) which reduces mitochondrial Ca2+ overload during myocardial ischemia, in digitonin-permeabilized H9c2 cells expressing the mitochondrial-targeted Ca2+ indicator, yellow cameleon 3.1. The elevation of mitochondrial Ca2+ concentration caused by an increase in extramitochondrial Ca2+ concentration was inhibited by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) or ruthenium red, but enhanced by CGP-37157, a mitochondrial NCX inhibitor. SEA0400 had no effect on mitochondrial Ca2+ under normal and ischemic conditions. Thus, the mitochondria-protective effects of SEA0400 could be explained by inhibition of plasmalemmal NCX but not mitochondrial NCX.

Knockdown of aquaporin-8 induces mitochondrial dysfunction in 3T3-L1 cells.

BACKGROUND: Aquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, H2O2 and NH3 has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression. METHODS: AQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements. RESULTS: In 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization. CONCLUSION: Mitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis. GENERAL SIGNIFICANCE: The AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.

Effect of terfenadine and pentamidine on the HERG channel and its intracellular trafficking: combined analysis with automated voltage clamp and confocal microscopy.

The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.

Blocking effect of NIP-142 on the KCNQ1/KCNE1 channel current expressed in HEK293 cells.

We examined the effect of NIP-142, a benzopyran compound with terminating effect on experimental atrial arrhythmia, on the KCNQ1/KCNE1 channel, which underlies the slow component of the cardiac delayed rectifier potassium channel (I(Ks)). NIP-142, as well as chromanol 293B, showed concentration-dependent blockade of the current expressed in HEK293 cells; the EC(50) value of NIP-142 and chromanol 293B for the inhibition of tail current was 13.2 µM and 4.9 µM, respectively. These results indicate that NIP-142 has blocking effect on the KCNQ1/KCNE1 channel current.

Effects of S(+)-efonidipine on the rabbit sinus node action potential and calcium channel subunits Ca(V)1.2, Ca(V)1.3 and Ca(V)3.1.

The effect of S(+)-efonidipine on sinus node action potential and calcium channel α-subunits was examined. The slope of the phase 4 depolarization of isolated rabbit sinus node tissue was significantly reduced by S(+)-efonidipine (1 µM), slightly reduced by nifedipine (1 µM), but was not affected by R(-)-efonidipine. S(+)-efonidipine (1 µM), inhibited the expressed Ca(V)1.2, Ca(V)1.3 and Ca(V)3.1 channel currents by 75.7%, 75.3% and 94.0%, nifedipine 84.0%, 43.2% and 14.9%, and R(-)-efonidipine 30.0%, 19.6% and 92.8%, respectively. Thus, the prolongation of the phase 4 depolarization of the rabbit sinus node by S(+)-efonidipine may be explained by blockade of the Ca(V)1.3 channel current.

Effect of NIP-142 on potassium channel alpha-subunits Kv1.5, Kv4.2 and Kv4.3, and mouse atrial repolarization.

Effects of NIP-142, a benzopyran compound which terminates experimental atrial arrhythmia, on potassium channel alpha-subunits and mouse atrial repolarization were examined. NIP-142 concentration-dependently blocked the outward current through potassium channel alpha subunits Kv1.5, Kv4.2 and Kv4.3 expressed in Xenopus oocytes. In isolated mouse atrial myocardia, NIP-142 prolonged the action potential duration and effective refractory period, and increased the contractile force. These results suggest that NIP-142 blocks the potassium channels underlying the transient and sustained outward currents, which may contribute to its antiarrhythmic activity.

Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.

Membrane-labeled MDCK cells and confocal microscopy for the analyses of cellular volume and morphology.

A clone of Madin-Darby canine kidney (MDCK) cells whose cell membrane was stably labeled with expressed cyan fluorescent protein (CFP) was established, and changes in their volume and shape induced by hyposmotic stress were analyzed with confocal microscopy. The membrane-targeted CFP was present not only on the cell membrane but also in the endoplasmic reticulum and Golgi apparatus, but was excluded from the mitochondria and cell nucleus. During hyposmosis, the initial swelling and the following regulatory volume decrease could be accurately measured by summation of the cellular volume in every confocal slice crossing the cell at different heights. Changes in the cellular height roughly paralleled the changes in cellular volume when the mean value was compared, but dissociation as much as 30% was observed for individual cells due to changes in cell shape. The present experimental system, which enables direct measurement of cell volume and simultaneous observation of various intracellular phenomena, would be useful for further investigation of cellular volume regulation.

Highly efficient in vivo gene transfection by plasmid/PEI complexes coated by anionic PEG derivatives bearing carboxyl groups and RGD peptide.

A new class of an anionic poly (ethylene glycol) derivative, PEG-Suc, bearing 17.7 pairs of carboxylic acid-side chains was synthesized. PEG-Suc deposited onto the DNA/polyethyleneimine complexes without destroying them even at high dose ratio. Coating of the DNA complexes by PEG-Suc recharged their surface to negative, and effectively protected them from the albumin-induced aggregation. Paired carboxyl groups in the side chains showed higher proton sponge effect. Negatively charged surface would diminish the electrostatic binding of the complexes to the cells, and the transfection efficiency on the cultured cells was not high. RGD peptide side chain as a ligand to malignant cell surfaces was then introduced to compensate the reduced electrical adhesion. RGD-PEG-Suc-coated plasmid/PEI complex brought about more than 3 times higher reporter protein activity on the cultured B16 cells. Those bio-compatible DNA complexes with ligand attained very high gene expression in tumor, lung, and liver after injection into mouse tail vein.

Involvement of intracellular Ca(2+) in the regulatory volume decrease after hyposmotic swelling in MDCK cells.

We examined the source of Ca(2+) involved in the volume regulation of Madin-Darby canine kidney (MDCK) cells with confocal microscopy and fluoroprobes. Hyposmosis induced a transient increase in cell volume, as well as cytoplasmic Ca(2+), which peaked at 3 to 5 min and gradually decreased to reach the initial value within about 30 min. This late decrease in cell volume, as well as the transient rise in cytoplasmic Ca(2+), was reduced in Ca(2+)-free solution and was abolished by pretreatment with thapsigargin. In conclusion, Ca(2+) released from the intracellular store contributes to the regulatory volume decrease following hyposmotic swelling in MDCK cells.

Involvement of the Na+/Ca2+ exchanger in ouabain-induced inotropy and arrhythmogenesis in guinea-pig myocardium as revealed by SEA0400.

Involvement of the Na+/Ca2+ exchanger in ouabain-induced inotropy and arrhythmogenesis was examined with a specific inhibitor, SEA0400. In right ventricular papillary muscle isolated from guinea-pig ventricle, 1 microM SEA0400, which specifically inhibits the Na+/Ca2+ exchanger by 80%, reduced the ouabain (1 microM)-induced positive inotropy by 40%, but had no effect on the inotropy induced by 100 microM isobutyl methylxantine. SEA0400 significantly inhibited the contracture induced by low Na+ solution. In HEK293 cells expressing the Na+/Ca2+ exchanger, 1 microM ouabain induced an increase in intracellular Ca2+, which was inhibited by SEA0400. The arrhythmic contractions induced by 3 microM ouabain were significantly reduced by SEA0400. These results provide pharmacological evidence that the Na+/Ca2+ exchanger is involved in ouabain-induced inotropy and arrhythmogenesis.

Quantitative fluorescence measurement of cardiac Na+/Ca2+ exchanger inhibition by kinetic analysis in stably transfected HEK293 cells.

We developed a method to quantitatively evaluate the potency of Na+/Ca2+ exchanger (NCX) inhibitors with fluorescence microscopy in NCX1-transfected HEK 293 cells. The reverse mode and forward mode NCX activities were measured as the ascending slope of the early phase increase in cytoplasmic Ca2+ concentration after change to low Na+ extracellular solution and the descending rate (inverse of the exponential time constant) on return to normal solution, respectively. Both modes of NCX were inhibited by SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) and KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), and the concentration-inhibition relationships for both inhibitors were in good agreement with those previously reported in voltage clamped cardiomyocytes.

Blockade by NIP-142, an antiarrhythmic agent, of carbachol-induced atrial action potential shortening and GIRK1/4 channel.

Mechanisms for the atria-specific action potential-prolonging action of NIP-142 ((3R*,4S*)-4-cyclopropylamino-3,4-dihydro-2,2-dimethyl-6-(4-methoxyphenylacetylamino)-7-nitro-2H-1-benzopyran-3-ol), a benzopyran compound that terminates experimental atrial arrhythmia, was examined. In isolated guinea-pig atrial tissue, NIP-142 reversed the shortening of action potential duration induced by either carbachol or adenosine. These effects were mimicked by tertiapin, but not by E-4031. NIP-142 concentration-dependently blocked the human G protein-coupled inwardly rectifying potassium channel current (GIRK1/4 channel current) expressed in HEK-293 cells with an EC50 value of 0.64 microM. At higher concentrations, NIP-142 blocked the human ether a go-go related gene (HERG) channel current with an EC50 value of 44 microM. In isolated guinea-pig papillary muscles, NIP-142 had no effect on the negative inotropic effect of carbachol under beta-adrenergic stimulation, indicating lack of effect on the muscarinic receptor and Gi protein. These results suggest that NIP-142 directly inhibits the acetylcholine-activated potassium current.

Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences.

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.

Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation.

Hyaluronic acid (HA), a natural anionic mucopolysaccharide, can be deposited onto the cationic surface of DNA/polyethyleneimine (PEI) complexes to recharge the surface potential and reduce nonspecific interactions with proteins. HA can also be used as a ligand to target specific cell receptors. Furthermore, HA-coating enhanced the transcriptional activity of the plasmid/PEI complexes, probably by loosening the tight binding between DNA and PEI, which facilitated the approach of transcription factors. Amphoteric HA derivative having spermine side chains (Spn-HA) with a structure similar to HMG protein showed higher transcription-enhancing activity than HA. Plasmid/PEI/Spn-HA ternary complex exhibited 29-fold higher transgene expression efficiency than naked plasmid/PEI complexes in CHO cells.

Enhancement of transcriptional activity of DNA complexes by amphoteric PEG derivative.

A water-soluble PEG derivative having both amino and carboxylic acid side chains (PEG-AC) was synthesized and explored for its transcription- and transfection-enhancing activity. PEG-AC could be deposited onto the surface of DNA/polycation complexes to form a ternary complex with slightly negative surface potential. PEG-AC-coating on the plasmid/PEI complexes obviously enhanced their transcriptional activity, and 31-fold higher consumption of UTP was observed. Amphoteric PEG-AC would loosen the tightly compacted DNA/PEI complex and facilitate the approach of transcriptional factors. PEG-AC also evidently improved the transgene expression level on the cultured CHO cells.

De-N-acetyllactotriaosylceramide as a novel cationic glycosphingolipid of bovine brain white matter: isolation and characterization.

A novel cationic lipid was separated from bovine brain white matter by a series of chromatographies on carboxymethyl-Sephadex and silica gel in chloroform and methanol. Its structure was identified unambiguously as de-N-acetyllactotriaosylceramide (deNAcLc(3)Cer) by mass spectrometry and (1)H NMR. The natural occurrence of this glycolipid in white matter extract was detected by immunostaining of thin-layer chromatography with monoclonal antibody 5F5, which is directed to deNAcLc(3)Cer and recognizes the terminal beta-glucosaminyl (GlcNH(2)) residue, having a free NH(2) group. A de-N-acetylase capable of hydrolyzing the N-acetyl group of Lc(3)Cer was detected in bovine brain extract using N-[(14)C]acetyl-labeled Lc(3)Cer as a substrate. The biogenesis and possible functional significance of deNAcLc(3)Cer are discussed.