Glucokinase is located in secretory granules of pancreatic D-cells

The effects of electrical stimulation, applied to the superior salivatory nucleus (SSN) or the cervical sympathetic nerve, on vascular permeability in nasal mucosa were studied in 16 cats. Plasma extravasation was quantified by using Evans blue. Vascular permeability in the cat nasal mucosa was increased by the electrical stimulation of SSN. Plasma extravasation induced by SSN stimulation was reduced by administration of nitric oxide synthase (NOS) antagonist, N(omega)-nitro-L-arginine methyl ester (L-NAME). Administration of atropine did not affect increased vascular permeability by SSN stimulation. We conclude that neurogenic plasma extravasation in cat nasal mucosa evoked by the parasympathetic nerve is not mediated by cholinergic fibers but rather by nitric oxide.     

Effects of oral vanadyl treatment on diabetes-induced alterations in the heart GLUT-4 transporter

Vanadyl sulfate was administered orally during a 10-week trial period to streptozotocin-diabetic and control male rats to test the hypothesis that chronic vanadyl supplementation would prevent the decline in cardiac muscle cell glucose transporter protein (GLUT-4) that otherwise manifests in conjunction with insulin deficiency. Isolated cardiac myocytes and cardiac sarcolemmal vesicles were prepared from heart tissue of rats that had been maintained on the following regimens: untreated control, oral vanadyl-supplemented control (0.6 mg/ml), untreated diabetic (streptozotocin-induced; 60 mg/kg), and vanadyl-supplemented diabetic. Myocytes isolated from untreated diabetic rat hearts had decreased rates of glucose oxidation. Chronic, oral administration of vanadyl to diabetic rats maintained glucose oxidation rates of cardiac myocytes at control levels. Immunoblot analyses revealed that total cardiac myocyte and sarcolemmal GLUT-4 glucose transporter protein levels were significantly lower in the diabetic group relative to control. Vanadyl treatment of diabetic rats produced a normalization of both sarcolemmal GLUT-4 and total cardiac myocyte levels towards control levels. The reduction of GLUT-4 mRNA levels seen with untreated diabetes was also completely prevented with vanadyl treatment. These results demonstrate that chronic-oral vanadyl sulfate supplementation limits the decline in glucose oxidative capacity of cardiac myocytes that otherwise manifests in the untreated diabetic state. This action of vanadyl may occur via a mechanism that is linked to the preservation of sarcolemmal GLUT-4 protein levels.     

A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a beta-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase. p27 exhibits isomerase activity with a kcat/Km of 0.18 microM-1 s-1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.     

Effects of space flight on GLUT-4 content in rat plantaris muscle

Administration of cisapride, 3 x 5 mg in a suspension one day before surgery and 30 mg 3 and 8 hours after abdominal surgery with subsequent administration of 2 x 30 mg in suppositories up to the time when oral ingestion is possible, hastens significantly the restoration of GIT motility as compared with placebo. It can be therefore recommended as effective medication in the prevention of complications caused by impaired motility of the digestive tract.     

Low-flow ischemia leads to translocation of canine heart GLUT-4 and GLUT-1 glucose transporters to the sarcolemma in vivo

BACKGROUND: Myocardial ischemia increases heart glucose utilization in vivo. However, whether low-flow ischemia leads to the translocation of glucose transporter (GLUT)-4 and/or GLUT-1 to the sarcolemma in vivo is unknown. METHODS AND RESULTS: In a canine model, we evaluated myocardial glucose metabolism in vivo and the distribution of GLUT-4 and GLUT-1 by use of immunoblotting of sarcolemma and intracellular membranes and immunofluorescence localization with confocal microscopy. In vivo glucose extraction increased fivefold (P < .001) and was associated with net lactate release in the ischemic region. Ischemia led to an increase in the sarcolemma content of both GLUT-4 (15 +/- 2% to 30 +/- 3%, P < .02) and GLUT-1 (41 +/- 4% to 58 +/- 3%, P < .03) compared with the nonischemic region and to a parallel decrease in their intracellular contents. Immunofluorescence demonstrated the presence of both GLUT-4 and GLUT-1 on cardiac myocytes. GLUT-1 had a more prominent cell surface pattern than GLUT-4, which was primarily intracellular in the nonischemic region. However, significant GLUT-4 surface labeling was found in the ischemic region. CONCLUSIONS: Translocation of the insulin-responsive GLUT-4 transporter from an intracellular storage pool to the sarcolemma occurs in vivo during acute low-flow ischemia. GLUT-1 is also present in an intracellular storage pool from which it undergoes translocation to the sarcolemma in response to ischemia. These results indicate that both GLUT-1 and GLUT-4 are important in ischemia-mediated myocardial glucose uptake in vivo.     

Type IB secretory phospholipase A2 is contained in insulin secretory granules of pancreatic islet beta-cells and is co-secreted with insulin from glucose-stimulated islets

People, now in their fifties and sixties, who were children during the Nazi Holocaust in WWII, endured persecution, massive traumatization, the constant risk of being killed, as well as the violent loss of (most of) their family members. They have internalized the resulting ongoing confusion and conflicts as to whether they should be alive or dead. This is maintained as an integral part of the child component of their compound personality, described in this paper. During the three years of the psychotherapy group, on which we focused here, these issues were expressed in different ways, such as suicide threats, occasional intolerance to physically remaining in the group, and outbursts of annihilating rage at the therapists. The confusion and conflicts about the legitimacy and risks of their survival came to a head during the termination process we insisted upon. Much attention has also been given to the intricacies of our countertransference--further complicated by our own connection to the Holocaust. We learned, and described, just how essential it is to acknowledge and process this countertransference in order to both contain the intense affects of anxiety, rage, and mourning in the groups, and enable them to be safely expressed. We imagine similar dynamics can be expected in group therapy with other populations who suffered massive, man-made traumatization.     

Changes in insulin-stimulated glucose transport and GLUT-4 protein in rat skeletal muscle after training

After running training, which increased GLUT-4 protein content in rat skeletal muscle by <40% compared with control rats, the training effect on insulin-stimulated maximal glucose transport (insulin responsiveness) in skeletal muscle was short lived (24 h). A recent study reported that GLUT-4 protein content in rat epitrochlearis muscle increased dramatically ( approximately 2-fold) after swimming training (J.-M. Ren, C. F. Semenkovich, E. A. Gulve, J. Gao, and J. O. Holloszy. J. Biol. Chem. 269, 14396-14401, 1994). Because GLUT-4 protein content is known to be closely related to skeletal muscle insulin responsiveness, we thought it possible that the training effect on insulin responsiveness may remain for >24 h after swimming training if GLUT-4 protein content decreases gradually from the relatively high level and still remains higher than control level for >24 h after swimming training. Therefore, we examined this possibility. Male Sprague-Dawley rats swam 2 h a day for 5 days with a weight equal to 2% of body mass. Approximately 18, 42, and 90 h after cessation of training, GLUT-4 protein concentration and 2-[1,2-3H]deoxy-D-glucose transport in the presence of a maximally stimulating concentration of insulin (2 mU/ml) were examined by using incubated epitrochlearis muscle preparation. Swimming training increased GLUT-4 protein concentration and insulin responsiveness by 87 and 85%, respectively, relative to age-matched controls when examined 18 h after training. Forty-two hours after training, GLUT-4 protein concentration and insulin responsiveness were still higher by 52 and 51%, respectively, in muscle from trained rats compared with control. GLUT-4 protein concentration and insulin responsiveness in trained muscle returned to sedentary control level within 90 h after training. We conclude that 1) the change in insulin responsiveness during detraining is directly related to muscle GLUT-4 protein content, and 2) consequently, the greater the increase in GLUT-4 protein content that is induced by training, the longer an effect on insulin responsiveness persists after the training.     

Glucose transporter expression in brain: relationship to cerebral glucose utilization

Glucose is the principle energy source for mammalian brain. Delivery of glucose from the blood to the brain requires its transport across the endothelial cells of the blood-brain barrier and across the plasma membranes of neurons and glia, which is mediated by the facilitative glucose transporter proteins. The two primary glucose transporter isoforms which function in cerebral glucose metabolism are GLUT1 and GLUT3. GLUT1 is the primary transporter in the blood-brain barrier, choroid plexus, ependyma, and glia; GLUT3 is the neuronal glucose transporter. The levels of expression of both transporters are regulated in concert with metabolic demand and regional rates of cerebral glucose utilization. We present several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. These include normal cerebral development in the rat, Alzheimer's disease, neuronal differentiation in vitro, and dehydration in the rat.     

Distribution of atrial natriuretic peptides in the sand rat (Psammomys obesus) in comparison to that in the rat

Atrial natriuretic peptides (ANP) are a family of humoral compounds involved in water and salt homeostasis. Immunoreactive ANP (IR-ANP) was determined in the plasma and tissues of the rat and the sand rat (Psammomys obesus) using sensitive and specific radioimmunoassay. IR-ANP from the rat and the sand rat elute at identical retention times from reverse phase HPLC indicating that the same chemical entity is present in both species. IR-ANP highest levels were found, in both species, in the heart but it was also present in the adrenal gland, lung, kidney, liver, plasma and several loci in the central nervous system. The IR-ANP levels in the heart, adrenal gland, kidney, liver, cerebellum and cerebral cortex were lower in the sand rat compared to the rat. The plasma IR-ANP level of the diabetes-resistant sand rat was further decreased to about 10% of the level in the diabetes-resistant sand rat.     

The vesicular monoamine transporter, in contrast to the dopamine transporter, is not altered by chronic cocaine self-administration in the rat

Although much evidence suggests that the brain dopamine transporter (DAT) is susceptible to dopaminergic regulation, only limited information is available for the vesicular monoamine transporter (VMAT2). In the present investigation, we used a chronic, unlimited-access, cocaine self-administration paradigm to determine whether brain levels of VMAT2, as estimated using [3H]dihydrotetrabenazine (DTBZ) binding, are altered by chronic exposure to a dopamine uptake blocker. Previously, we showed that striatal and nucleus accumbens DAT levels, as estimated by [3H]WIN 35,428 and [3H]GBR 12,935 binding, are altered markedly using this animal model (Wilson et al., 1994). However, in sequential sections from the same animals, [3H]DTBZ binding was normal throughout the entire rostrocaudal extent of the basal ganglia (including striatum and nucleus accumbens), cerebral cortex, and diencephalon, as well as in midbrain and brainstem monoamine cell body regions, both on the last day of cocaine access and after 3 weeks of drug withdrawal. These data provide additional evidence that VMAT2, unlike DAT, is resistant to dopaminergic regulation.     

The GM2-1 ganglioside islet autoantigen in insulin-dependent diabetes mellitus is expressed in secretory granules and is not beta-cell specific

The pancreatic islet monosialo-ganglioside (GM2-1), an autoantigen in insulin-dependent diabetes mellitus (IDDM) recently shown to be the target of autoantibodies associated with diabetes development in relatives of IDDM patients, is islet specific within the pancreas, and its expression is metabolically regulatable. In the present study we sought to establish 1) whether GM2-1 is beta-cell specific, and 2) its intracellular localization. To this end, we analyzed the pattern of ganglioside expression in highly purified beta- and non-beta-cells isolated from rat islets. In addition, ganglioside levels were determined in subcellular fractions of a rat beta-cell line (INS). No qualitative or quantitative difference was found in the pattern of ganglioside expression between beta and non-beta rat islet cells, with GM3, GM2-1, and GD3 gangliosides expressed in both cell populations. Within INS cells, GM2-1 ganglioside was expressed in the fraction containing secretory granules and, to a lesser extent, in plasma membranes; GM3 was expressed in secretory granules, whereas GD3 was found only in plasma membranes. These data indicate that the GM2-1 autoantigen is not beta-cell specific within the islets, in accordance with the observation that this molecule is a target of islet cell autoantibodies that bind to the whole pancreatic islet. Interestingly, this autoantigen is present in secretory granules similarly to other autoantigens in IDDM (insulin, carboxypeptidase H, 38-kDa protein, etc.), suggesting that the autoimmunity to the components of this organelle may be central to the pathogenesis of the disease.     

Specific interaction between erythrocytes and a glucose-carrying polymer mediated by the type-1 glucose transporter (GLUT-1) on the cell membrane

A reducing glucose-carrying polymer, called poly [3-O-(4'-vinylbenzyl)-D-glucose] (PVG), interacted with erythrocytes carrying the type-1 glucose transporter (GLUT-1) on the cell membrane. The cooperative interaction between a number of GLUT-1s and a number of reducing 3-O-methyl-D-glucose moieties on a PVG polymer chain is responsible for the increase in the interaction with erythrocytes. In contrast to the PVG homopolymer, other sugar-carrying polymers showed lower interaction with erythrocytes. The affinity of erythrocytes and PVG was studied using FITC-labeled glycopolymers. The fluorescence intensity significantly changed, whereas a small change in fluorescence intensity was observed for other homopolymers. The specific interaction between GLUT-1 on erythrocytes and the PVG polymer carrying reducing glucose was suppressed by the inhibitors, phloretin, phloridzin, and cytochalasin B, and a monoclonal antibody to GLUT-1. Direct observation by confocal laser microscopy with the use of FITC-labeled PVG demonstrated that erythrocytes interacted with the soluble form of the PVG polymer via GLUT-1, while fluorescence labeling of the cell surface was prevented on pretreatment with the monoclonal antibody to GLUT-1.     

Divergence of beta-myosin heavy chain (betaMHC) expression in fetal rat cardiomyocytes in vitro and adult rat heart in vivo

The myosin heavy chain gene products are an important determinant of myocardial functional properties. Although a strong positive element (beta f1) within the betaMHC promoter region has previously been identified, to date no species comparisons in promoter strength have been made. To examine this question, we have used betaMHC deletion constructs, containing the rat or human beta f1 enhancer region, to determine expression both in vitro using rat fetal cardiomyocytes and in vivo by direct injection into adult rat heart. When reporter constructs were transfected into cultured fetal rat cardiomyocytes, the human beta reporter was expressed more than 3 fold above the equivalent rat construct. Exchange of the beta f1 enhancer indicated that the human sequence of the beta f1 enhancer was largely responsible. However, these findings were not replicated when the reporters were injected into the adult rat heart. In the adult myocardium the levels of reporter expression were similar for the betaMHC promoter reporters studied. These findings demonstrate a divergence between primary cardiomyocytes maintained in culture and the cardiomyocytes found within the intact adult heart.     

The bactericidal/permeability-increasing protein (BPI) is present in specific granules of human eosinophils

Eosinophils participate in the inflammatory response seen in allergy and parasitic infestation, but a role in host defense against bacterial infection is not settled. The bactericidal/permeability-increasing protein (BPI) has been demonstrated in neutrophils and it exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. Using the Western blot technique, a 55-kD band, corresponding to BPI, was detected in lysates from both neutrophils and eosinophils. The localization of BPI in immature and mature eosinophils was investigated using immunoelectron microscopy. BPI was found in immature and mature specific granules of eosinophils and was detected in phagosomes as well, indicating release of the protein from the granules into the phagosomes. Using a specific enzyme-linked immunosorbent assay, eosinophils were shown to contain 179 ng of BPI/5 x 10(6) eosinophils compared with 710 ng BPI/5 x 10(6) neutrophils. The presence of BPI in eosinophils suggests a role for these cells in host defense against Gram-negative bacterial invasion or may suggest a role for BPI against parasitic infestation.     

Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.     

Glucose transporter levels in a male spontaneous non-insulin-dependent diabetes mellitus rat of the Otsuka Long-Evans Tokushima Fatty strain

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a new strain of spontaneous non-insulin-dependent diabetes mellitus (NIDDM) models. To evaluate the role of glucose transporters (GLUT) in the development of diabetes in this model, we examined the action of insulin on the translocation of GLUT4 and GLUT1 in isolated adipocytes, and the GLUT4 protein levels in muscles. Long-Evans Tokushima Otsuka (LETO) rats were used as a control strain. In adipocytes, the GLUT4 protein levels in OLETF rats at 30 weeks of age (diabetic stage) were considerably lower than those in LETO rats at the same age. At a pre-diabetic stage (7 weeks), there were no significant differences in GLUT4 protein levels in adipocytes between LETO and OLETF rats. However, the degree of GLUT4 translocation in OLETF rats was lower than that in LETO rats at 7 weeks of age. There were no differences in GLUT1 levels in adipocytes between the two strains. In muscles, the decrease in GLUT4 protein was observed in OLETF rats at 30 weeks of age. Whether such a difference is under the influence of hyperglycemia was also examined using rats rendered diabetic by 70% pancreatectomy. OLETF rats aged 7 weeks were subjected to partial pancreatectomy (Px) and sham pancreatectomy (sham). At 4 weeks after surgery, GLUT4 protein levels in adipose tissues and skeletal muscles were determined. GLUT4 decrease was observed for both tissues of hyperglycemic Px rats compared with euglycemic sham. Moreover, we examined the direct effect of glucose on GLUT4 protein using primary cultured adipocytes of OLETF rats at 5 weeks of age. After 7-day culture with normal (5.6 mmol/l) or high (25 mmol/l) concentrations of glucose, the GLUT4 protein levels in adipocytes decreased at 25 mmol/l glucose compared with 5.6 mmol/l glucose. These findings suggest an early defect in the insulin resistance of OLETF rats probably reflects impaired GLUT4 translocation. The GLUT4 decrease, which occurs later in the process appears to be a consequence, rather than a cause of diabetes in OLETF rats.     

Arachidonic acid activates a proton current in the rat glutamate transporter EAAT4

The excitatory amino acid transporter EAAT4 is expressed predominantly in Purkinje neurons in the rat cerebellum (1-3), and it participates in postsynaptic reuptake of glutamate released at the climbing fiber synapse (4). Transporter-mediated currents in Purkinje neurons are increased more than 3-fold by arachidonic acid, a second messenger that is liberated following depolarization-induced Ca2+ activation of phospholipase A2 (5). In this study we demonstrate that application of arachidonic acid to oocytes expressing rat EAAT4 increased glutamate-induced currents to a similar extent. However, arachidonic acid did not cause an increase in the rate of glutamate transport or in the chloride current associated with glutamate transport but rather activated a proton-selective conductance. These data reveal a novel action of arachidonate on a glutamate transporter and suggest a mechanism by which synaptic activity may decrease intracellular pH in neurons where this transporter is localized.     

Mechanism of block by 4-aminopyridine of the transient outward current in rat ventricular cardiomyocytes

Binocular information has been shown to be important for the programming and control of reaching and grasping. But even without binocular vision, people are still able to reach out and pick up objects accurately - albeit less efficiently. As part of a continuing investigation into the role that monocular cues play in visuomotor control, we examined whether or not subjects could use retinal motion information, derived from movements of the head, to help program and control reaching and grasping movements when binocular vision is denied. Subjects reached out in the dark to an illuminated sphere presented at eye-level, under both monocular and binocular viewing conditions with their head either free to move or restrained. When subjects viewed the display monocularly, they showed fewer on-line corrections when they were allowed to move their head. No such difference in performance was seen when subjects were allowed a full binocular view. This study, combined with previous work with neurological patients, confirms that the visuomotor system "prefers" to use binocular vision but, when this information is not available, can fall back on other monocular depth cues, such as information produced by motion of the object (and the scene) on the retina, to help program and control manual prehension.