Alpha-SNAP functions in insulin exocytosis from mature, but not immature secretory granules in pancreatic beta cells.

To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively.

Functional role of arg-gly-asp (RGD)-binding sites on beta1 integrin in embryo implantation using mouse blastocysts and human decidua.

Amino acid residues 140-164 of integrin beta1 comprise an Arg-Gly-Asp (RGD) cross-linking region. The present study was undertaken to study the role of the RGD cross-linking region of integrin beta1 subunit in embryo implantation. Decidual cells attached to fibronectin (FN)-coated dishes.

Glucose metabolism in cholinoceptive cortical rat brain regions after basal forebrain cholinergic lesion.

To address the question whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease are interrelated with, or consequences of, basal forebrain cholinergic cell loss, an experimental approach was employed to produce cortical cholinergic dysfunction in rat brain by administration of the cholinergic immunotoxin 192IgG-saporin. [14C]D-glucose utilization in brain homogenates, D-glucose-displaceable [3H]cytochalasin B binding to glucose transporters (GLUT). Northern and Western analyses, as well as in vivo [14C]2-deoxyglucose autoradiography were used to quantify the regional glucose metabolism.

Colocalization of GLUT3 and choline acetyltransferase immunoreactivity in the rat retina.

Toward elucidating the functional aspects ofGLUT3, a primary neuronal glucose transporter isoform in the vertebrate central nervous system, this study examined its expression in cholinergic amacrine cells made identifiable by the presence of acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), in the rat retina. Double-immunofluorescence staining of adult rat retinal tissue with anti-GLUT3 and anti-ChAT antibodies revealed characteristic stratified GLUT3 immunoreactivity (GLUT3-IR) in the inner plexiform layer (IPL) that was identical to the arborization pattern of ChAT-positive neuronal processes there. In addition, approximately 30-50% of intensely GLUT3-immunoreactive cell bodies in the inner nuclear layer and ganglion cell layer showed ChAT-IR, while the majority of ChAT-positive cell bodies were also intensely GLUT3 immunoreactive.

alpha-soluble N-ethylmaleimide-sensitive factor attachment protein is expressed in pancreatic beta cells and functions in insulin but not gamma-aminobutyric acid secretion.

The function of soluble N-ethylmaleimide-sensitive attachment protein-alpha (alpha-SNAP) in exocytosis still remains obscure. This study was conducted to determine the physiological role of alpha-SNAP in the secretion of insulin and gamma-aminobutryric acid (GABA) from pancreatic beta cells. Reverse transcriptase-polymerase chain reaction analysis of total RNA isolated from rat islets disclosed alpha-SNAP, but not beta-SNAP, mRNA expression, and an immunofluorescence study of rat pancreas showed that alpha-SNAP was present predominantly in the cytoplasm of the islets of Langerhans.

Functional role of focal adhesion kinase in the process of implantation.

The expression and function of focal adhesion kinase (FAK) in human decidual cells were investigated. This kinase is localized to focal adhesions in fibroblasts, and is phosphorylated on tyrosine in normal and src-transformed fibroblasts. Immunofluorescent staining revealed that the cultured decidual cells expressed high levels of FAK at the cell periphery.

Syntaxin, but not soluble NSF attachment protein (SNAP), biosynthesis by rat pancreatic islets is regulated by glucose in parallel with proinsulin biosynthesis.

Recent studies have revealed that soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE)-related proteins, originally identified in neural tissues, are also expressed in pancreatic beta cells. In this study, we investigated the effect of glucose on syntaxin 1 and alpha/beta SNAP biosynthesis in pancreatic beta cells and we demonstrated that syntaxin 1, but not alpha/beta SNAP biosynthesis by rat isolated pancreatic islets was stimulated specifically by glucose nearly in parallel with proinsulin biosynthesis. Stimulation of syntaxin 1 and proinsulin biosynthesis by glucose was dose-dependent (Km = approximately 8 mmol/l) and reached the maximum (about 8-12 fold) at concentrations over 11 mmol/l.

Non-functional role of syntaxin 2 in insulin exocytosis by pancreatic beta cells.

This study was designed in order to examine the expression and functional role of syntaxin 2/epimorphin in pancreatic beta cells. Northern blot analysis revealed that syntaxin 2 mRNA was able to be detected in mouse beta TC3 cells, but not in isolated mouse islets. In agreement with this result, immunoblot analysis detected an appreciable amount of syntaxin 2 protein in beta TC3 cells, but not in mouse islets.

Overexpressed syntaxin 1A/HPC-1 inhibits insulin secretion via a regulated pathway, but does not influence glucose metabolism and intracellular Ca2+ in insulinoma cell line beta TC3 cells.

We have previously established a stable beta TC3 cell line that overexpresses syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin.

Rat C6 glioma cell growth is related to glucose transport and metabolism.

In order to establish whether growth of glioma cells is associated with glucose transport and metabolism, we investigated expression of the glucose transporter and hexokinase, as well as glucose transport and glucose phosphorylation in rat C6 glioma cells growing at different rates. Rat C6 glioma cells were subcloned to produce four different cell lines (CL1, CL2, CL3 and CL4) differing in growth, differentiation and morphology: CL1 cells were slow-growing with an astrocytic appearance whereas CL4 cells grew rapidly and were small and spindle-shaped. Immunocytochemical analysis using glial fibrillary acidic protein and galactocerebroside antibodies revealed that CL1 and CL4 cells differentiate to astrocytes and oligodendrocytes respectively.

Effects of insulin-like growth factor-I on follicle growth, oocyte maturation, and ovarian steroidogenesis and plasminogen activator activity in the rabbit.

We examined the effects of insulin-like growth factor (IGF)-I on follicular growth, oocyte maturation, and ovarian steroidogenesis and plasminogen activator (PA) activity in vitro, using a perfused rabbit ovary preparation in order to determine whether the follicle-stimulating effects of growth hormone (GH) are mediated by IGF-I. The addition of IGF-I to the perfusate stimulated follicular growth and the resumption of meiosis in follicular oocytes in a dose-dependent manner. There was no significant difference in the production of progesterone by perfused rabbit ovaries between IGF-I-treated and control ovaries, whereas IGF-I increased the production of estradiol (E2) by perfused rabbit ovaries in a dose-dependent manner.

Expression of glucose transporters in rat brain following transient focal ischemic injury.

We have investigated the serial changes in the transcription and translation of the rat glucose transporter (GLUT) 1 and 3 genes after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. Northern blot analysis and in situ hybridization study were performed to determine the chronological change and regional expression. In the ipsilateral anterior cerebral artery (ACA) cortex, GLUT1 mRNA expression was increased at 12 h (11.

Localization and ontogeny of GLUT3 expression in the rat retina.

This study investigates the presence, localization, and developmental expression of a neuron-specific facilitated-diffusion glucose transporter, GLUT3, in the rat retina so as to elucidate molecular mechanisms regulating glucose homeostasis in support of the visual function. Immunoblot analysis using anti-GLUT3 antibody (ALM3-C) revealed the presence of GLUT3 as a heterogeneously glycosylated protein with an average molecular weight of approximately 44 kDa. Although immunofluorescence staining showed it to be localized primarily in the inner and outer plexiform layers, some of the cell bodies in the inner nuclear layer also showed weak immunoreactivity.

Function of beta 1 integrins on human decidual cells during implantation.

In the present study, the effects of antibodies against specific beta 1 integrin heterodimers on mouse embryo attachment and spreading were tested to identify the role of the beta 1 integrins in early implantation. We developed assays for the attachment of mouse embryos and for trophoblastic spreading on cultured human decidual cells. Blastocysts became attached to the cultured decidual cells in the presence of a purified mouse monoclonal IgG1 antibody (negative control) after the embryos hatched from the zona pellucida.

Expression of beta 1 integrins in human endometrial stromal and decidual cells.

The present study was undertaken to investigate the expression of beta1 integrins in human endometrium and decidua using flow cytometry, immunohistochemistry, and immunoprecipitation. Fluorescence-activated flow cytometry demonstrated the greater expression of the beta 1, alpha 1, alpha 2, and alpha 5 subunits of the beta1 integrin family in cultured stromal cells from the midsecretory phase, than in those of the early proliferative phase. The addition of estradiol (E2) and progesterone (P) to cultured stromal cells in the early proliferative phase increased the expression of beta1 integrins in vitro.

Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor.

Background: Oct-3, a pluripotent cell-specific POU transcription factor, appears to be a key regulator in pluripotential early embryonic cells and germ cells. In order to study how pluripotency is maintained, it is essential to know what genes are regulated by Oct-3.

Results: By employing a subtraction method, we identified several pluripotent cell-specific genes.

Insulin-like growth factor binding protein-3 inhibits gonadotropin-induced ovulation, oocyte maturation, and steroidogenesis in rabbit ovary.

The effects of insulin-like growth factor binding proteins (IGFBPs) on human CG (hCG)-induced oocyte maturation, ovulation, steroidogenesis, and intrafollicular plasminogen activator (PA) activity were investigated in rabbit ovaries perfused in vitro. The addition of IGFBP-3, but not IGFBP-1, to the perfusate dose dependently inhibited hCG-induced ovulation, whereas ovulation failed to occur in any ovaries perfused with medium or IGFBP-3 alone. IGFBP-3 (100 ng/ml) significantly inhibited the resumption of meiosis in ovulated ova and follicular oocytes in hCG-treated ovaries, as well as the hCG-stimulated production of estradiol (E2), but not progesterone, by the perfused ovaries.

Expression and functional role of syntaxin 1/HPC-1 in pancreatic beta cells. Syntaxin 1A, but not 1B, plays a negative role in regulatory insulin release pathway.

Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated syntaxin 1 expression in pancreatic beta cells and the functional role of syntaxin 1 in the insulin release mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure.

Ethanol decreases the levels of GLUT2 glucose transporter mRNA in hepatocytes.

In order to elucidate the implication of GLUT2 in the impaired glucose metabolism caused by ethanol, we examined if ethanol affects GLUT2 glucose transporter mRNA expression in the liver. After the adult rats were fed with and/or without ethanol for 1 or 4 weeks, hepatocytes were isolated and total RNA was extracted from them. The levels of GLUT2 mRNA in hepatocytes isolated from 1 week-ethanol fed rats estimated by Northern blot analysis did not change compared to those of untreated rats, those levels in hepatocytes from 4 weeks-ethanol fed rats decreased markedly.

Glucose transporter expression and functional role of hexokinase in insulin biosynthesis in mouse beta TC3 cells.

It was previously reported that insulin biosynthesis in mouse beta TC3 cells was regulated by glucose (Nagamatsu, S., and D. F.

Abundant expression of GLUT1 and GLUT3 in rat embryo during the early organogenesis period.

The developmental change of both GLUT1 and GLUT3 protein in rat embryonal and fetal brain was examined using Western blot analysis and immunohistochemistry. The brains were collected from fetuses (gestational days 10 to 20), newborn, and adult rats. On day 10, the levels of GLUT1 and GLUT3 expressions were twofold higher than those of adult levels, but thereafter decreased rapidly as the gestation progressed.

Effects of beta-1 integrins in the process of implantation.

The expression and function of beta 1 integrins on human decidual cells were investigated. Flow cytometric analysis revealed that the cultured decidual cells expressed a high level of the beta 1 subunit on the cell surface. Mouse blastocysts attached to and spread onto cultured human decidual cells.

GLUT2 expression in the rat retina: localization at the apical ends of Müller cells.

In order to understand the molecular basis of glucose regulation supporting visual function, this study examined the presence of GLUT2, a facilitated-diffusion glucose transporter isoform, and delineated its localization in the rat retina. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated the presence of GLUT2 mRNA, and immunoblot analysis using polyclonal antibody specific to rat GLUT2 revealed a band at a molecular weight of approximately 60 kDa, indicating the presence of GLUT2 protein in the rat retina. Fluorescence and electron microscopy localized GLUT2 expression to the apical ends of Müller cells that face the inter-photoreceptor space.

Existence of an enzymatic pathway furnishing arachidonic acid for prostaglandin synthesis from arachidonoyl CoA in rabbit kidney medulla.

We examined whether arachidonoyl CoA (AA-CoA) can be a possible supplier of arachidonic acid (AA) for prostaglandin (PG) synthesis in rabbit kidney medulla. AA-CoA was preincubated with or without the 105,000 g supernatant (cytosol) fraction from the kidney medulla for 5 min at 37 degrees C followed by the incubation with the microsomal fraction (0.5 mg protein) (a rich source of PG synthesizing enzymes) in the presence of reduced glutathione and hydroquinone for 5 min at 37 degrees C, and the formed PGE2, F2 alpha and D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization.

Developmental expression of GLUT3 glucose transporter in the rat brain.

The ontogeny of the GLUT3 glucose transporter gene and protein expression was studied in rat brain. Northern blot analysis using total RNA from rat brains at different developmental stages revealed that the levels of GLUT3 mRNA were very low during the embryonic stage and increased towards the postnatal stage. Immunohistochemistry using a specific antibody showed that the expression of GLUT3 protein was barely detectable in the embryonic stage, but was clearly detected on the plasma membrane of neuronal cells from 10 days after birth to the adult.