Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.     

Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.     

Inversely Estimating the Vertical Profile of the Soil CO2 Production Rate in a Deciduous Broadleaf Forest Using a Particle Filtering Method

Carbon dioxide (CO₂) efflux from the soil surface, which is a major source of CO₂ from terrestrial ecosystems, represents the total CO₂ production at all soil depths. Although many studies have estimated the vertical profile of the CO₂ production rate, one of the difficulties in estimating the vertical profile is measuring diffusion coefficients of CO₂ at all soil depths in a nondestructive manner. In this study, we estimated the temporal variation in the vertical profile of the CO₂ production rate using a data assimilation method, the particle filtering method, in which the diffusion coefficients of CO₂ were simultaneously estimated. The CO₂ concentrations at several soil depths and CO₂ efflux from the soil surface (only during the snow-free period) were measured at two points in a broadleaf forest in Japan, and the data were assimilated into a simple model including a diffusion equation. We found that there were large variations in the pattern of the vertical profile of the CO₂ production rate between experiment sites: the peak CO₂ production rate was at soil depths around 10 cm during the snow-free period at one site, but the peak was at the soil surface at the other site. Using this method to estimate the CO₂ production rate during snow-cover periods allowed us to estimate CO₂ efflux during that period as well. We estimated that the CO₂ efflux during the snow-cover period (about half the year) accounted for around 13% of the annual CO₂ efflux at this site. Although the method proposed in this study does not ensure the validity of the estimated diffusion coefficients and CO₂ production rates, the method enables us to more closely approach the “actual” values by decreasing the variance of the posterior distribution of the values.     

Involvement of selective epithelial cell death in the formation of feather buds on a bioengineered skin

Selective cell death by apoptosis plays important roles in organogenesis. Apoptotic cells are observed in the developmental and homeostatic processes of several ectodermal organs, such as hairs, feathers, and mammary glands. In chick feather development, apoptotic events have been observed during feather morphogenesis, but have not been investigated during early feather bud formation. Previously, we have reported a method for generating feather buds on a bioengineered skin from dissociated skin epithelial and mesenchymal cells in three-dimensional culture. During the development of the bioengineered skin, epithelial cavity formation by apoptosis was observed in the epithelial tissue. In this study, we examined the selective epithelial cell death during the bioengineered skin development. Histological analyses suggest that the selective epithelial cell death in the bioengineered skin was induced by caspase-3-related apoptosis. The formation of feather buds of the bioengineered skin was disturbed by the treatment with a pan-caspase inhibitor. The pan-caspase inhibitor treatment suppressed the rearrangement of the epithelial layer and the formation of dermal condensation, which are thought to be essential step to form feather buds. The suppression of the formation of feather buds on the pan-caspase inhibitor-treated skin was partially compensated by the addition of a GSK-3β inhibitor, which activates Wnt/β-catenin signaling. These results suggest that the epithelial cell death is involved in the formation of feather buds of the bioengineered skin. These observations also suggest that caspase activities and Wnt/β-catenin signaling may contribute to the formation of epithelial and mesenchymal components in the bioengineered skin.     

A furostanol glucuronide from Solanum lyratum

A new furostanol glucuronide and three known glycosides, SL-O, aspidistrin and methyl proto-aspidistrin, were isolated from the fresh immature berries of Solanum lyratum. The structure of the new compound was characterized as 26-O-β-D-glucopyranosyl-(22ξ,25R-3β,22,26-trihydroxyfurost-5-ene 3-O-α-L-rhamnopyranosyl-(1 → 2)-[β-D-glucopyranosyl-(1 → 3)]-β-D-glucuronopyranoside.     

Increased calcium release from sarcoplasmic reticulum stimulated by inositol trisphosphate in spontaneously hypertensive rat heart cells

It is known that inositol (1, 4, 5)-trisphosphate (IP₃) stimulates Ca²⁺ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP₃ on Ca²⁺ release from SR in cardiac myocytes. The effect of IP₃ on Ca²⁺ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP₃ on Ca²⁺ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP₃ on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with⁴⁵Ca²+, and then incubated with the indicated concentrations of IP³ for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca²⁺ release stimulated by 10 M (1, 4, 5)-IP₃ was detected by fura-2. In⁴⁵Ca²⁺ prelabeled SR, the maximal Ca²⁺ release was achieved at 10 M IP₃ incubated for 1 min. The release of Ca²⁺ was higher in Sr of SHR than in the SR of WKY. IP₃ stimulates Ca²⁺ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.     

Cranial cartilages: Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular

The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage-related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage-related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural-crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural-crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study.