Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.

Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.

Development of nobiletin-methyl hesperidin amorphous solid dispersion: Novel application of methyl hesperidin as an excipient for hot-melt extrusion.

A novel amorphous solid dispersion (ASD) of poorly water-soluble nobiletin (Nob) with highly water-soluble methyl hesperidin (MeHes) was developed. Mixtures of Nob and excipients (MeHes, cellulose derivatives, and synthetic polymers) were processed by hot-melt extrusion (HME). Powder X-ray diffraction analysis proved that most of the HME products were fully amorphized. In dissolution studies, Nob-MeHes ASD showed a prominently higher Nob concentration than other HME products with polymeric excipients. Nob concentration upon dissolution of Nob-MeHes ASD was 400 and 7.5 times higher than that upon dissolution of crystalline Nob and a Nob-MeHes physical mixture, respectively. In addition, Nob-MeHes ASD showed good preservation stability for 6 months under an accelerated condition of 40 °C and 80% relative humidity. Permeation studies using a Caco-2 cell monolayer showed that Nob-MeHes ASD markedly increased the amount of Nob transported. In mice, the plasma Nob concentration and accumulated amount of Nob in various tissues drastically increased after administration of Nob-MeHes ASD. This is the first successful application of MeHes, with a relatively low glass-transition temperature, as an excipient for an ASD formulation prepared by hot-melt extrusion. The drastic improvement in Nob concentration with a small-molecule excipient may be an important finding.

Preferential Adsorption of l-Histidine onto DOPC/Sphingomyelin/3ß-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol Liposomes in the Presence of Chiral Organic Acids.

We investigated the effect of organic acids such as mandelic acid (MA) and tartaric acid (TA) on the adsorption behavior of both histidine (His) and propranolol (PPL) onto liposomes. A cationic and heterogeneous liposome prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM)/3ß-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Ch) in a ratio of (4/3/3) showed the highest adsorption efficiency of MA and TA independent of chirality, while neutral liposome DOPC/SM/cholesterol = (4/3/3) showed low efficiency. As expected, electrostatic interactions were dominant in MA or TA adsorption onto DOPC/SM/DC-Ch = (4/3/3) liposomes, suggesting that organic acids had adsorbed onto SM/DC-Ch-enriched domains. The adsorption behaviors of organic acids onto DOPC/SM/DC-Ch = (4/3/3) were governed by Langmuir adsorption isotherms. For adsorption, the membrane polarities slightly decreased (i.e., membrane surface was hydrophilic), but no alterations in membrane fluidity were observed. In the presence of organic acids that had been preincubated with DOPC/SM/DC-Ch = (4/3/3), the adsorption of l- and d-His onto those liposomes was examined. Preferential l-His adsorption was dramatically prevented only in the presence of l-MA, suggesting that the adsorption sites for l-His and l-MA on DOPC/SM/DC-Ch = (4/3/3) liposomes are competitive, while those for l-His and d-MA, l-TA, and d-TA are isolated.

Effect of Boundary Edge in DOPC/DPPC/Cholesterol Liposomes on Acceleration of l-Histidine Preferential Adsorption.

In order to investigate the interaction of hydrophilic molecules with liposomal membranes, we employed 1-(4-(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl) as fluorescent probes to monitor the surface regions of the membrane, and the results for various liposomes were plotted in correlation diagrams. According to the formation of a variety of phase states, different tendencies of decreasing surface hydrophobicity were observed in the liposomes that were modified with high concentrations of cholesterol or in the liposomes that were composed of ternary components. These liposomes, with hydrophobic surfaces, also showed preferential adsorption of l-histidine (l-His), and the hydrophobicity of the liposomal membrane at the surface changed during l-His adsorption regardless of the initial liposomal properties. Furthermore, we revealed that accelerated adsorption of l-His and preferential binding was induced in ternary liposomes forming boundaries between two separate phases.

Chiral Selective Adsorption of Ibuprofen on a Liposome Membrane.

We investigated the key factors that affect enantioselective adsorption of ibuprofen (IBU) on a liposome membrane by changing its lipid composition: the liposome membrane shows different membrane fluidity, surface charge, content of chiral components, and heterogeneity (nanodomain). Nonspecific interactions (hydrophobic and electrostatic) were revealed to be an important factor in enhancing the adsorbed amount of IBU, based on adsorption experiments carried out using single lipids (DPPC, DMPC, DOPC, and DLPC) and positively charged liposomes (DOTAP and liposome containing DC-Ch). Furthermore, control of the boundary edge (i.e., the nanodomain size) derived from the membrane heterogeneity was important for enantioselective adsorption; as well as multiple weak interactions between lipid molecules and IBU enantiomers. The above findings provided a good index for constructing liposomal chiral adsorbents.

Liposome Membrane as a Platform for the L-Pro-Catalyzed Michael Addition of trans-ß-Nitrostyrene and Acetone.

Herein, we show that the L-proline (L-Pro)-catalyzed Michael addition of trans-ß-nitrostyrene and acetone can proceed in "water" using liposome membranes and that the membrane fluidity and polarity are major controlling factors for this reaction. The highest conversion and rate constant of the reaction within the liposomes was achieved with the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-3-trimethylammoniumpropane (DPTAP) system. The catalytic activity of L-Pro in the liposome suspension was found to be comparable to that in a DMSO system. The reaction rate constant was found to be controlled by both the phase state of the liposome membrane and the surface charge on the membrane. Greater enantioselectivity was achieved in the presence of the liposomes than in DMSO solution, with corresponding enantiomeric excess values of 97.6% for the DOPC/DPTAP liposome system and 10% in DMSO. The hydrophobic region of the liposome membrane, which is a relatively stable self-organizing system, can serve as an effective "platform" for molecular recognition and selective conversion in aqueous media.

High performance optical resolution with liposome immobilized hydrogel.

We prepared liposome immobilized hydrogels (LI-gels) for analysis and separation of chiral molecules, to overcome the drawbacks of liposomes such as low stability, and difficulties with handling and isolation from sample solutions. The amounts of liposomes in the hydrogels were larger than those in other solid matrices reported previously. The liposome morphology was intact, and its original properties, such as fluidity and phase transition behaviors, were preserved. We investigated the chiral recognition performance of the LI-gel, as described in our previous paper. Our results indicate that the enantioselectivity of the LI-gel was higher than those of conventional methods and of the liposomes alone. Our prepared LI-gel therefore overcomes the drawbacks of liposomes, and has potential applications in analysis and separation, including chiral separation.

Chiral Recognition of L-Amino Acids on Liposomes Prepared with L-Phospholipid.

In this study, we demonstrated that liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) can recognize several l-amino acids, but not their d-enantiomers, by analyzing their adsorptive behavior and using circular dichroism spectroscopy. Changes in liposomal membrane properties, determined based on fluorescent probe analysis and differential scanning calorimetry, were induced by l-amino acid binding. UV resonance Raman spectroscopy analysis suggested that the chiral recognition was mediated by electrostatic, hydrophobic, and hydrogen bond interactions, where the recognition site could therefore be constructed on the DPPC membrane. Our findings clearly indicate the potential function of liposomes in asymmetric recognition.

Microfluidic platforms with monolithically integrated hierarchical apertures for the facile and rapid formation of cargo-carrying vesicles.

We fabricated a simple yet robust microfluidic platform with monolithically integrated hierarchical apertures. This platform showed efficient diffusive mixing of the introduced lipids through approximately 8000 divisions with tiny pores (~5 µm in diameter), resulting in massive, real-time production of various cargo-carrying particles via multi-hydrodynamic focusing.