Overexpression of Slc22a18 facilitates fat accumulation in mice.

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Epigallocatechin-3-gallate is an inhibitor of Na+, K(+)-ATPase by favoring the E1 conformation.

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na(+),K(+)-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC(50) values of about 1 microM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E1P and E2P toward E(1)P, and reduced the rate of the E1P to E2P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H+, K(+)-ATPase and sarcoplasmic reticulum Ca(2+)-ATPase) as well as the Na(+),K(+)-ATPase, whereas soluble ATPases (bacterial F(1)-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na(+),K(+)-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC50 to 10 microM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.

Isolation of stable (alphabeta)4-Tetraprotomer from Na+/K+-ATPase solubilized in the presence of short-chain fatty acids.

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) >> Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.

Na+- and K+-dependent oligomeric interconversion among alphabeta-protomers, diprotomers and higher oligomers in solubilized Na+/K+-ATPase.

Protein fractions of a higher-oligomer (H), (alphabeta)(2)-diprotomer (D) and alphabeta-protomer (P) were separated from dog kidney Na(+)/K(+)-ATPase solubilized in the presence of NaCl and KCl. Na(+)/K(+)-dependent interconversion of the oligomers was analysed using HPLC at 0 degrees C. With increasing KCl concentrations, the content or amount of D increased from 27.6 to 54.3% of total protein, i.e. DeltaC(max) = 26.7%. DeltaC(max) for the sum of D and H was equivalent to the absolute value of DeltaC(max) for P, regardless of the anion present, indicating that K(+) induced the conversion of P into D and/or H, and Na(+) had the opposite effect. When enzymes that had been denatured to varying degrees by aging were solubilized, DeltaC(max) increased linearly with the remaining ATPase activity. The magnitude of the interconversion could be explained based on an equilibrium of D <==> 2P, assuming 50-fold difference in the K(d) between KCl and NaCl, and coexistence of unconvertible oligomers, which comprised as much as 39% of the eluted protein. Oligomeric interconversion, determined as a function of the KCl or NaCl concentration, showed K(0.5)s of 64.8 microM and 6.50 mM for KCl and NaCl, respectively, implying that oligomeric interconversion was coupled with Na(+)/K(+)-binding to their active transport sites.