Structure-dependent inhibitory effects of green tea catechins on insulin secretion from pancreatic ß-cells.

The effects of green tea catechins on glucose-stimulated insulin secretion (GSIS) were investigated in the ß-cell line INS-1D. Epigallocatechin gallate (EGCG) at 10 µM or gallocatechin gallate (GCG) at 30 µM caused significant inhibitory effects on GSIS, and each of these at 100 µM almost abolished it. In contrast, epicatechin (EC) or catechin (CA) had no effect on GSIS at concentrations up to 100 µM. We thus investigated the structure-activity relationship by using epigallocatechin (EGC) and gallocatechin (GC) containing a trihydroxyl group in the B-ring, and epicatechin gallate (ECG) and catechin gallate (CG) containing the gallate moiety. EGC, GC, and ECG caused an inhibition of GSIS, although significant effects were obtained only at 100 µM. At this concentration, EGC almost abolished GSIS, whereas GC and ECG partially inhibited it. In contrast, CG did not affect GSIS at concentrations up to 100 µM. EGCG also abolished the insulin secretion induced by tolbutamide, an ATP-sensitive K(+) channel blocker, and partially inhibited that induced by 30 mM K(+). Moreover, EGCG, but not EC, inhibited the oscillation of intracellular Ca(2+) concentration induced by 11.1 mM glucose. These results suggest that some catechins at supraphysiological concentrations have inhibitory effects on GSIS, the potency of which depends on their structure; the order of potency was EGCG>GCG>EGC>GC≈ECG. The inhibitory effects seem to be mediated by the inhibition of voltage-dependent Ca(2+) channels, which is caused, at least in part, by membrane hyperpolarization resulting from the activation of K(+) channels.

Analysis of localization and reorganization of germ plasm in Xenopus transgenic line with fluorescence-labeled mitochondria.

Germ plasm is found in germ-line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria-line). Germ plasm with EGFP-labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ-line cells in Dria-line females. Using the Dria-line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.

Periocular injection of in situ hydrogels containing Leu-Ile, an inducer for neurotrophic factors, promotes retinal ganglion cell survival after optic nerve injury.

Intraocular administration of neurotrophic factors has been shown to delay irreversible degeneration of retinal ganglion cells (RGCs). It would be beneficial for the treatment of optic nerve (ON) injury if such neurotrophic factors could be delivered in a less-invasive manner. The dipeptide leucine-isoleucine (Leu-Ile) appears to induce the production of neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), in the brain. We therefore administered Leu-Ile via periocular depot injection in rats and investigated the dipeptide's ability to induce BDNF and GDNF in the retina and to delay RGC loss in an ON injury model. Poloxamer-alginate hydrogels containing Leu-Ile were injected into the subconjunctival space of intact or ON-injured rats. BDNF and GDNF levels in the retina were determined by an enzyme immunoassay. Survival of RGCs was assessed in retinal flatmounts. Activation of extracellular signal-regulated kinases (ERK) and cAMP response element binding protein (CREB) in the retina was examined by Western blotting. At 2 h after injection of fluorescein isothiocyanate-conjugated Leu-Ile, the fluorescence intensities in the retina were 4.3-fold higher than those in the saline control. Treatment with Leu-Ile significantly increased the retinal levels of BDNF at 6 h and GDNF at 6-72 h after injection. Treatment with Leu-Ile significantly increased RGC survival to 14 days after ON injury and enhanced the activation of ERK at 72 h and CREB at 48 h after injection in the ON-injured retina. These results suggest that periocular delivery of Leu-Ile induces BDNF and GDNF production in the retina, which may eventually enhance RGC survival after ON injury.

Involvement of stretch-activated cation channels in hypotonically induced insulin secretion in rat pancreatic beta-cells.

In isolated rat pancreatic beta-cells, hypotonic stimulation elicited an increase in cytosolic Ca(2+) concentration ([Ca(2+)](c)) at 2.8 mM glucose. The hypotonically induced [Ca(2+)](c) elevation was significantly suppressed by nicardipine, a voltage-dependent Ca(2+) channel blocker, and by Gd(3+), amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca(2+)](c) elevation was not inhibited by suramin, a P(2) purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of beta-cells and produced outwardly rectifying cation currents; Gd(3+) inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd(3+) significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic beta-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca(2+) channels and thus elevating insulin secretion.