The action mechanism of zinc(II) complexes with insulinomimetic activity in rat adipocytes

Zinc (Zn), an essential trace element, and its complexes have recently been known to exhibit insulinomimetic activities. However, the action mechanism of Zn(II) has yet been obscure. The purpose of the present study was to estimate the action mechanism of the Zn(II) complexes. We found first that Zn given in the chemical forms such as Zn(maltolate)2 and Zn(threoninate)2 complexes is highly uptaken in the isolated rat adipocytes compared with that of Zn(picolinate)2. Then, the action mechanism for the insulinomimetic activities was examined in terms of free fatty acid release from the adipocytes. Four Zn(II) compounds, ZnSO4, Zn(picolinate)2, Zn(maltolate)2, and Zn(threoninate)2, inhibited the free fatty acid release from the adipocytes treated with epinephrine (adrenaline). By using several inhibitors for fatty acids and glucose metabolisms in the adipocytes, the following results were obtained. (1) Zn(picolinic acid)2 complex acts on the insulin receptor and PI3-k, which relate to the glucose uptake, as indicated by the experiments using hydroxy-2-naphthalenylmethyl phosphonic acid tris acetoxy methyl ester (HNMPA-(AM)3) and wortmannin, respectively. (2) ZnSO4, and Zn(maltolate)2 and Zn(threoninate)2 complexes affect a glucose transporter 4 (GLUT 4), which is involved in the glucose uptake as indicated by the results using cytochalasin B. (3) Four Zn(II) compounds affect the activation of the phosphodiesterase as indicated by the experiments using cilostamide. These results indicate that the Zn(II) compounds promote the glucose uptake into the adipocytes by affecting at least three sites in the adipocytes, which in turn normalize the blood glucose levels in the experimental diabetic animals.     

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip-Syntaxin4 interaction.     

Docosahexaenoic acid provides protection from impairment of learning ability in Alzheimer's disease model rats

Docosahexaenoic acid (C22:6, n-3), a major n-3 fatty acid of the brain, has been implicated in restoration and enhancement of memory-related functions. Because Alzheimer's disease impairs memory, and infusion of amyloid-beta (Abeta) peptide (1-40) into the rat cerebral ventricle reduces learning ability, we investigated the effect of dietary pre-administration of docosahexaenoic acid on avoidance learning ability in Abeta peptide-produced Alzheimer's disease model rats. After a mini-osmotic pump filled with Abeta peptide or vehicle was implanted in docosahexaenoic acid-fed and control rats, they were subjected to an active avoidance task in a shuttle avoidance system apparatus. Pre-administration of docosahexaenoic acid had a profoundly beneficial effect on the decline in avoidance learning ability in the Alzheimer's disease model rats, associated with an increase in the cortico-hippocampal docosahexaenoic acid/arachidonic acid molar ratio, and a decrease in neuronal apoptotic products. Docosahexaenoic acid pre-administration furthermore increased cortico-hippocampal reduced glutathione levels and glutathione reductase activity, and suppressed the increase in lipid peroxide and reactive oxygen species levels in the cerebral cortex and hippocampus of the Alzheimer's disease model rats, suggesting an increase in antioxidative defence. Docosahexaenoic acid is thus a possible prophylactic means for preventing the learning deficiencies of Alzheimer's disease.     

Avian Podocytes,Which Lack Nephrin,Use Adherens Junction Proteins at Intercellular Junctions

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal—where AJ proteins are suppressed to an extremely low level—and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.     

Choline availability modulates human neuroblastoma cell proliferation and alters the methylation of the promoter region of the cyclin-dependent kinase inhibitor 3 gene

Choline is an important methyl donor and a component of membrane phospholipids. In this study, we tested the hypothesis that choline availability can modulate cell proliferation and the methylation of genes that regulate cell cycling. In several other model systems, hypomethylation of cytosine bases that are followed by a guanosine (CpG) sites in the promoter region of a gene is associated with increased gene expression. We found that in choline-deficient IMR-32 neuroblastoma cells, the promoter of the cyclin-dependent kinase inhibitor 3 gene (CDKN3) was hypomethylated. This change was associated with increased expression of CDKN3 and increased levels of its gene product, kinase-associated phosphatase (KAP), which inhibits the G(1)/S transition of the cell cycle by dephosphorylating cyclin-dependent kinases. Choline deficiency also reduced global DNA methylation. The percentage of cells that accumulated bromodeoxyuridine (proportional to cell proliferation) was 1.8 times lower in the choline-deficient cells than in the control cells. Phosphorylated retinoblastoma (p110) levels were 3 times lower in the choline-deficient cells than in control cells. These findings suggest that the mechanism whereby choline deficiency inhibits cell proliferation involves hypomethylation of key genes regulating cell cycling. This may be a mechanism for our previously reported observation that stem cell proliferation in hippocampus neuroepithelium is decreased in choline-deficient rat and mouse fetuses.     

Structural analysis and taste evaluation of γ-glutamyl peptides comprising sulfur-containing amino acids

The structures, flavor-modifying effects, and CaSR activities of γ-glutamyl peptides comprising sulfur-containing amino acids were investigated. The chemical structures, including the linkage mode of the N-terminal glutamic acid, of γ-L-glutamyl-S-(2-propenyl)-L-cysteine (γ-L-glutamyl-S-allyl-L-cysteine) and its sulfoxide isolated from garlic were established by comparing their NMR spectra with those of authentic peptides prepared using chemical methods. Mass spectrometric analysis also enabled determination of the linkage modes in the glutamyl dipeptides by their characteristic fragmentation. In sensory evaluation, these peptides exhibited flavor-modifying effects (continuity) in umami solutions less pronounced but similar to that of glutathione. Furthermore, the peptides exhibited intrinsic flavor due to the sulfur-containing structure, which may be partially responsible for their flavor-modifying effects. In CaSR assays, γ-L-glutamyl-S-methyl-L-cysteinylglycine was most active, which indicates that the presence of a medium-sized aliphatic substituent at the second amino acid residue in γ-glutamyl peptides enhances CaSR activity.     

Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers

The cell permeability of hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, hesperetin permeated cells in the apical-to-basolateral direction at the rate (Jap-->bl) of 10.43+/-0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023+/-0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of hesperetin was almost constant irrespective of the TER. Apically loaded NaN3 or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap-->bl of hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap-->bl and Jbl-->ap of hesperetin were almost the same (5.75+/-0.40 and 5.16+/-0.73 nmol/min/mg protein). Jbl-->ap of hesperetin in the presence of a proton gradient was lower than Jbl-->ap in the absence of a proton gradient. Furthermore, Jbl-->ap in the presence of a proton gradient remarkably increased upon addition of NaN3 specifically to the apical side. These results indicate that hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of hesperetin, that is proton-coupled and energy-dependent polarized transport.     

The role of gibberellins in the growth of floral organs of Pharbitis nil

Evidence that the synthesis of GA₃ is involved in the growthof floral orga'ns of Pharbitis nil is presented. GAs in floralorgans at different developmental stages were surveyed usingTLC followed by the bioassay with two dwarf rice seedlings,‘Tanginbozu’ and ‘Waito-C’. The amountof GAs in the petal and stamen increased rapidly after the petalemerged from calyx, reached a maximum 12 hr before anthesis,then declined markedly thereafter. The GA content in the calyxremained unchanged before and after anthesis, and that in thepistil increased after anthesis. Pharbitis flowers containedat least two active GAs, one of which was probably GA₃, theother appeared to be GA₁₉. GA₃ was detected in relatively largeamounts in both the petal and stamen during their rapid elongation.In the calyx, which showed little increase in fresh weight duringrapid flower growth, GA₉ was the dominant GA. Exogenously suppliedGA₃ promoted elongation of sections in excised young filaments.Sucrose was necessary for definite growth promotion by GA₃.GA₁₉ had little effect on filament elongation, and IAA was ratherinhibitive. (Received July 29, 1972; )