Neuromodulatory effects of hydrogen peroxide on central neurons in the feeding network of the mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

Hydrogen peroxide at a concentration of 100 μM was found to exert a pronounced modulatory effect on motor (R/L cells in B1–B4 clusters) and modulatory (R/L cerebral giant cells) neurons in the feeding neural network of the mollusc Lymnaea stagnalis as manifested in changes in the firing rate, membrane potential level and spike amplitude in these cells. The observed effects were reversible, transient, and reached their peak values in 1 min since application of the preparation. Injection of hydrogen peroxide into the cavity of the cephalopedal sinus resulted in no statistically significant changes in the parameters of mollusc feeding behavior. Hydrogen peroxide is assumed to act as a rapid neuromodulator towards neurons of the central feeding rhythm generator in Lymnaea stagnalis.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

Octopamine: a new feeding modulator in Lymnaea

The role of octopamine (OA) in the feeding system of the pond snail, Lymnaea stagnalis, was studied by applying behavioural tests on intact animals, and a combination of electrophysiological analysis and morphological labelling in the isolated central nervous system. OA antagonists phentolamine, demethylchlordimeform (DCDM) and 2-chloro-4-methyl-2-(phenylimino)-imidazolidine (NC-7) were injected into intact snails and the sucrose-induced feeding response of animals was monitored. Snails that received 25 to 50 mg kg^-1 phentolamine did not start feeding in sucrose, and the same dose of NC-7 reduced the number of feeding animals by 80 to 90% 1 to 3 hours after injection. DCDM treatment reduced feeding by 20 to 60%. In addition, both phentolamine and NC-7 significantly decreased the feeding rate of those animals that still accepted food after 1 to 6 hours of injection. In the central nervous system a pair of buccal neurons was identified by electrophysiological and morphological criteria. After double labelling (intracellular staining with Lucifer yellow followed by OA-immunocytochemistry) these neurons were shown to be OA immunoreactive, and electrophysiological experiments confirmed that they are members of the buccal feeding system. Therefore the newly identified buccal neurons were called OC neurons (putative octopamine containing neurons or octopaminergic cells). Synchronous intracellular recordings demonstrated that the OC neurons share a common rhythm with feeding neurons either appearing spontaneously or evoked by intracellularly stimulated feeding interneurons. OC neurons also have synaptic connections with identified members of the feeding network: electrical coupling was demonstrated between OC neurons and members of the B4 cluster motoneurons, furthermore, chemically transmitted synaptic responses were recorded both on feeding motoneurons (B1, B2 cells) and the SO modulatory interneuron after the stimulation of OC neurons. However, elementary synaptic potentials could not be recorded on the follower cells of OC neurons. Prolonged (20 to 30 s) intracellular stimulation of OC cells activated the buccal feeding neurons leading to rhythmic activity pattern (fictive feeding) in a way similar to OA applied by perfusion onto isolated central nervous system (CNS) preparations. Our results suggest that OA acts as a modulatory substance in the feeding system of Lymnaea stagnalis and the newly identified pair of OC neurons belongs to the buccal feeding network.     

D-Glucose but Not Insulin Reduces N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-Induced Shape Changes in Suspended Human Neutrophils

The effects of glucose (5–25 mM) and insulin concentration (40–320 U/ml) on the cell shape of neutrophil granulocytes from healthy humans were studied. Both non-activated and N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-activated neutrophils in suspension were used as a model for initial chemotactic activation of neutrophil locomotion. D-glucose, but not the non-metabolizable analogue 3-O-methyl-D-glucose, dose-dependently reduced the fMet-Leu-Phe-induced (10^–8M) neutrophil elongation. Insulin, either alone or in combination with 25 mM D-glucose, was without effect on the fMet-Leu-Phe-induced neutrophil elongation. Furthermore, the inhibitory effect of D-glucose was observed already after 1 min of exposure to D-glucose and fMet-Leu-Phe. D-glucose diminished the fraction of neutrophils with elongated locomotor shape by changing it into an irregular cell shape, suggesting that at least part of the D-glucose effect could be associated with mechanisms determining the typical locomotor shape. The present results suggest that D-glucose through its metabolism, but without the involvement of insulin, reduces chemotactically induced elongation to a locomotor neutrophil shape, and thus neutrophil motility, and that this effect of glucose appears prior to adhesion. This glucose-induced inhibition of the neutrophil chemotactic response may be involved in the neutrophil deficiency seen in diabetes mellitus.     

Stimulative effects of insulin on Toxoplasma gondii replication in 3T3-L1 cells

The influence of insulin and 2-deoxy-glucose (D-glucose) on the intracellular protozoan Toxoplasma gondii replication in 3T3-L1 cells was investigated. Insulin and D-glucose had a dose-responsive mitogenic effect on intracellular T. gondii replication and development in 3T3-L1 cells. Insulin concentrations between 10(-2) and 10(-1) microg/ml combination of 4.5 g/l D-glucose in DMEM medium gave maximum stimulus to T. gondii replication. The number of tachyzoites increased rapidly, with the growth peaking typically on day 3 or 4 of culture, and then declining quickly. However, insulin, in the absence of d-glucose, had comparably less effect on T. gondii growth than two of their combination. d-glucose concentrations significantly affected the tachyzoite replication and appear to be indispensable for maintaining the host 3T3-L1 cells.     

Dopamine-containing substantia nigra units are unresponsive to changes in plasma glucose levels induced by dietary factors, glucose infusions or insulin administration in freely moving cats

Dopamine-containing neurons in the pars compacta of the substantia nigra showed no significant change in activity during 48 hours of food deprivation in cats that were maintained on either a high carbohydrate diet or a low carbohydrate-high protein diet. Plasma glucose levels declined significantly during this time period in the high carbohydrate diet group, and increased slightly in the low carbohydrate-high protein diet group. In addition, there was no significant change in the activity of dopaminergic neurons in food deprived cats during feeding behavior, during which glucose levels were restored to normal. Intravenous infusion of glucose in freely moving cats, which elevated plasma glucose levels from 82 to 719 mg/100 ml and midbrain glucose from 4.3 to 12.2 mumoles/g, was also without effect on the activity of dopaminergic neurons. Insulin administration to cats maintained on a diet of standard cat chow and fasted for 18 hours decreased plasma and brain glucose to 32.8 mg/100 ml and 2.1 mumoles/g, respectively, but, again, there was no significant change in nigral unit activity. These data demonstrate that central dopaminergic neurons are unresponsive to fluctuations in brain and plasma glucose, and argue against a role for central dopamine systems in the regulation of feeding behavior and energy metabolism.     

The human red cell glucose transporter in octyl glucoside. High specific activity of monomers in the presence of membrane lipids

Human red cell membranes were stripped of peripheral proteins and partially solubilized with 50-260 mM octyl glucoside at 2-14 mg protein/ml, to find conditions that afford a high concentration of active glucose transporter after purification on DEAE-cellulose. Transporter-egg yolk phospholipid vesicles were prepared by gel filtration. The specific D-glucose equilibrium exchange activities increased with increasing dilution of the glucose transporter. At 260 mM octyl glucoside the glucose transporter became partially denaturated. At 225 mM detergent the DEAE-cellulose chromatography showed one main and one minor fraction of active glucose transporter. Nucleoside transport activity was enriched in the minor fraction. Solubilization with 75 mM octyl glucoside at 8 mg protein/ml gave a maximal concentration of purified transporter, 0.8 mg/ml, probably corresponding to complete solubilization. The phospholipids were partially retarded on the DEAE-cellulose. The specific D-glucose equilibrium exchange was high, up to 200 nmol glucose/micrograms transporter in two min at 50 mM glucose. High performance gel filtration in octyl glucoside indicated that the transporter formed dimers during the fractionation. These eluted at Mr 125,000, partially separated from the phospholipids, which appeared at Mr 55,000 (cf. Mascher, E. and Lundahl, P. (1987) J. Chromatogr. 397, 175-186). The D-glucose transport activity was low in the main fraction and high in the transporter-phospholipid fraction. Mixing of these fractions did not increase the activity. The glucose transporter is probably dependent on one or more specific membrane lipid(s). Presumably the transporter dimerizes and loses activity upon removal of these lipids.     

Estimation of the yield coefficient of Pseudomonas sp. strain DP-4 with a low substrate (2,4-dichlorophenol [DCP]) concentration in a mineral medium from which uncharacterized organic compounds were eliminated by a non-DCP-degrading organism

The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2, 4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 microg of DCP-C ml(-1). A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 microg of C ml of DCP(-1) and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 microg of C ml(-1) was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 microg of C ml(-1) was not degraded in mixed culture even by a high density, 10(5) CFU ml(-1), of DP-4. When glucose was added to this mixed culture to a final concentration of 1 microg of C ml(-1), the initial concentration of 0.01 microg of C ml of DCP(-1) was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 microg of C ml(-1). The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 microg of C ml of DCP(-1) was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.     

Comparative pharmacology of feeding in molluscs

1. This paper reviews the role of transmitters in identified neurons of gastropod molluscs in generating and modulating fictive feeding. 2. In Lymnaea and Helisoma the 3 phase rhythm is generated by sets of interneurons which use acetylcholine for the N1 (protraction) phase, glutamate for the N2 (rasp) phase interneurons. The N3 interneurons are likely to use several different transmitters, of which one is octopamine. 3. In all the species examined, serotonin (5-HT) is released from giant cerebral cells. Other amines, including dopamine and octopamine, are present in the buccal ganglia and all these amines activate or enhance feeding. 4. Nitric oxide (NO), mostly originating from sensory processes, can also activate fictive feeding, but (at least in Lymnaea) may also be released centrally from buccal (B2) and cerebral neurons (CGC). 5. The central pattern generator for feeding is also modulated by peptides including APGWamide, SCP(B) and FMRFamide. 6. There is increasing evidence that most of these transmitters/modulators act on feeding neurons through second messenger systems--allowing them to act as longer-lasting neuromodulators of the feeding network. 7. Many of the transmitters are used in similar ways by each of the gastropods examined so far, so that their function in the CNS seems to have been conserved through evolution.     

Responses of thoracic spinal neurons to activation and desensitization of cardiac TRPV1-containing afferents in rats

The purpose of this study was to examine how upper thoracic spinal neurons responded to activation and desensitization of cardiac transient receptor potential vanilloid-1 (TRPV1)-containing afferent fibers. Extracellular potentials of single T3 spinal neurons were recorded in pentobarbital-anesthetized, paralyzed, and ventilated male rats. To activate cardiac nociceptive receptors, a catheter was placed in the pericardial sac to administer various chemicals: bradykinin (BK; 10 microg/ml, 0.2 ml), capsaicin (CAP, 10 microg/ml, 0.2 ml), or a mixture of algesic chemicals (AC; 0.2 ml) containing adenosine 10(-3) M, BK, serotonin, histamine, and PGE(2), 10(-5) M for each. Spinal neurons that responded to intrapericardial BK and/or CAP were used in this study. Results showed that 81% (35/43) of the neurons had excitatory responses to both intrapericardial BK and CAP, and the remainder responded to either BK or CAP. Intrapericardial resiniferatoxin (RTX) (0.2 microg/ml, 0.2 ml, 1 min), which desensitizes TRPV1-containing nerve endings, abolished excitatory responses to both BK (n = 8) and CAP (n = 7), and to AC (n = 5) but not to somatic stimuli. Intrapericardial capsazepine (1 mg/ml, 0.2 ml, 3 min), a specific antagonist of TRPV1, sharply attenuated excitatory responses to CAP in 5/5 neurons, but responses to BK in 5/5 neurons was maintained. Additionally, intrapericardial capsazepine had no significant effect on excitatory responses to AC in 3/3 neurons. These data indicated that intrapericardial BK-initiated spinal neuronal responses were linked to cardiac TRPV1-containing afferent fibers, but were not dependent on TRPV1. Intraspinal signaling for cardiac nociception was mediated through CAP-sensitive afferent fibers innervating the heart.     

Expression of glucose transporter GLUT3 by endotoxin in cultured rat astrocytes: the role of nitric oxide

The induction of nitric oxide (NO) synthase in astrocytes by endotoxin and/or cytokine treatment is associated with increased glucose consumption and glycolysis, but the mechanism whereby this phenomenon occurs remains obscure. In this work, we have addressed this issue and found that incubation of cultured rat astrocytes with lipopolysaccharide (LPS; 1 microg/mL) for 24 h increased the level of constitutively expressed GLUT1 glucose transporter mRNA, and triggered GLUT3 mRNA expression, which was absent in normal astrocytes. The occurrence of GLUT3 protein after LPS treatment was corroborated by western blotting and immunocytochemistry. A 4-h incubation of astrocytes in the absence of glucose, or under an oxygen-poor (3%) atmosphere also resulted in GLUT3 mRNA overexpression. Experiments performed with 2-deoxy-D-[U-14C]glucose (at 0.1 mM of D-glucose) confirmed that LPS (0.1-10 microg/mL) dose-dependently increased the rate of glucose uptake (by a factor of 1.6 at 1 microg/mL of LPS), which was paralleled with the increase in NO synthesis. Furthermore, blockade of NO synthase with 2-amino-5,6-dihydro-6-methyl-(4H)-1,3-thiazine (AMT; 50 microM) partially (by 45%) prevented the LPS-mediated increase in glucose uptake. Finally, incubation of astrocytes with the NO donor 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA; 100 microM) increased by a factor of 1.4 the rate of glucose uptake. We conclude that the increase in GLUT3-driven glucose uptake in astrocytes would have a neuroprotective role under conditions in which NO formation is combined with hypoglycaemia, such as in brain ischemia.     

Regulation of MAL1+ gene expression encoding maltase in Schizosaccharomyces pombe by added inositol

In this study, the effects of inositol addition on maltase activity and expression of MAL1+ gene encoding maltase in Schizosaccharomyces pombe were investigated. The maximum specific maltase activity was observed, when the concentration of inositol reached 6.0 microg/ml in the synthetic medium containing 2.0% glucose. At 1.0 microg/ml inositol concentration, the maltase activity continuously decreased, as initial glucose concentration was higher than 0.1%. mRNA encoding maltase and phosphatidylinositol (PI) content were higher in the cells grown in the synthetic medium with 6.0 microg/ml of inositol and 2.0% glucose than those with 1.0 microg/ml of inositol. These results demonstrated that higher inositol concentration in the synthetic medium could derepress MAL1+ gene expression in S. pombe and PI might be involved in derepression of MAL1+ gene expression in S. pombe probably by PI-type signalling pathway.     

Anomeric specificity of D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.     

Anomeric specificity of D-glucose production by rat hepatocytes

The anomeric specificity of D-glucose metabolism in intact hepatocytes remains a matter of debate. This issue was further investigated in the present study, which is based on the quantification of the alpha- and beta-anomers of the 13C-enriched isotopomers of D-glucose generated by rat liver cells exposed to either D-[1-13C] fructose or D-[2-13C] fructose in the presence of D2O. The D-[1-13C]glucose/D-[6-13C]glucose paired ratios found in the cells exposed to D-[1-13C] fructose and the D-[2-13C]glucose/D-[5-13C]glucose paired ratios found in the cells exposed to D-[2-13C] fructose yielded a paired beta/alpha ratio averaging (mean +/- S.E.M.) 79.3 +/- 6.1%. In the case of the isotopomers of D-glucose formed by gluconeogenesis, the D-[2-13C]glucose/D-[5-13C]glucose and D-[3-13C]glucose/D-[4-13C]glucose paired ratios found in cells exposed to D-[1-13C] fructose, as well as the D-[1-13C]glucose/D-[6-13C]glucose and D-[3-13C]glucose/D-[4-13C]glucose paired ratios found in cells exposed to D-[2-13C]fructose, yielded an alpha/beta paired ratio averaging 75.0 +/- 5.8%. Last, in the cells exposed to D-[2-13C]fructose, the beta/alpha ratio for the C2-deuterated isotopomers of D-[2-13C]glucose represented 78.9 +/- 3.7% of that for the C5-deuterated isotopomers of D-[5-13C]glucose. The three values representative of the anomeric specificity of D-glucose production by liver cells were not significantly different from one another, with an overall mean value of 76.9 +/- 3.6%. These findings unambiguously document that the anomeric specificity of phosphoglucoisomerase is operative in intact hepatocytes, resulting in a preferential output of the alpha-anomer of 13C-enriched D-glucose under the present experimental conditions.     

The facilitation of defensive reactions during food consumption in the snail helix: the participation of glucose and gastrin/cholecystokinin-like peptide]

Molecular and cellular mechanisms of the interrelations between the feeding and defense behaviour were studied in a snail Helix lucorum. The dynamics of defense reactions was investigated in snails with different levels of feeding motivation. Defense reactions were suppressed in hungry snails, while 15-20 min after the beginning of food intake they were facilitated. The facilitation depended on a duration of starvation. Injection of 0.5 ml of 5 mM glucose solution (up to the glucose level in the haemolymph of a food satiated snail, 1.6-2.0 mM) or injections of 20-30 ng of synthetic analogues of the gastrointestinal peptides (pentagastrin of octapeptide cholecystokinin, CCK-8) facilitated the defense reaction in a hungry snail. Parameters of the facilitation were similar to those in the period of food intake. Activity of the command neurons of defense behaviour (L-PPL1) after the carrot juice application to the lip of a semi-intact preparation from a hungry snail was glucose-dependent. Similar glucose-dependent changes of L-PPL1 activity were found after CCK-8, but not FMRFamide application during the perfusion with 0.5 mM glucose. L-PPL1, but not L-PPa2-3 neurons were most sensitive to glucose and CCK-8 level changes in the Ringer solution. Adaptive significance of the behavioural phenomena as well as glucose and gastrin/CCK-like peptide participation in these processes are discussed.     

Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis

The electrically coupled giant neurosecretory neurons VD1 and RPD2 of Lymnaea stagnalis were found to have coupling coefficients ranging from ca. 0.1-0.6. When the fluoroescent dye Lucifer Yellow was injected intracellularly into one of the neurons, in most preparations no dye was observed to pass through into the coupled cell body or the process leading to it. There was no apparent correlation between the amount of dye coupling and the length of time allowed for diffusion of the dye in the cells. In eight preparations, the electrical coupling coefficient was measured before dye was injected. There was no correlation between dye coupling and the electrical coupling coefficient.     

Role of leptin in the control of postprandial pancreatic enzyme secretion.

Leptin released by adipocytes has been implicated in the control of food intake but recent detection of specific leptin receptors in the pancreas suggests that this peptide may also play some role in the modulation of pancreatic function. This study was undertaken to examine the effect of exogenous leptin on pancreatic enzyme secretion in vitro using isolated pancreatic acini, or in vivo in conscious rats with chronic pancreatic fistulae. Leptin plasma level was measured by radioimmunoassay following leptin administration to the animals. Intraperitoneal (i.p.) administration of leptin (0.1, 1, 5, 10, 20 or 50 microg/kg), failed to affect significantly basal secretion of pancreatic protein, but markedly reduced that stimulated by feeding. The strongest inhibition has been observed at dose of 10 microg/kg of leptin. Under basal conditions plasma leptin level averaged about 0.15 +/- 0.04 ng/ml and was increased by feeding up to 1.8 +/- 0.4 ng/ml. Administration of leptin dose-dependently augmented this plasma leptin level, reaching about 0.65 +/- 0.04 ng/ml at dose of 10 microg/kg of leptin. This dose of leptin completely abolished increase of pancreatic protein output produced by ordinary feeding, sham feeding or by diversion of pancreatic juice to the exterior. Leptin (10(-10)-10(-7) M) also dose-dependently attenuated caerulein-induced amylase release from isolated pancreatic acini, whereas basal enzyme secretion was unaffected. We conclude that leptin could take a part in the inhibition of postprandial pancreatic secretion and this effect could be related, at least in part, to the direct action of this peptide on pancreatic acini.     

Studies on the energy metabolism of opossum (Didelphis Virginiana) erythrocytes. I. Utilization of carbohydrates and purine nucleosides

Opossum erythrocytes filtered through cellulose columns were used to estimate their permeability to D-glucose and optimum inorganic phosphate requirement for D-glucose utilization at pH 7.4 and 8.1. D-Glucose readily penetrated opossum red cells; there was no measurable difference whether plasma or electrolyte solution served as the suspending medium. Optimum extracellular inorganic phosphate concentration for glucose utilization as indicated by red cell lactate production was pH-dependent, with a sharp optimum of 30 mmol/liter at pH 8.1. Whereas glucose, fructose, mannose, dihydroxyacetone, adenosine, and inosine were readily utilized at pH 7.4 and Pi 30 mmol/liter as shown by net lactate and ATP production by the red cells, galactose and ribose as substrates were not metabolized. In electrolyte, Pi 30 mmol/liter, and pH 7.4 glucose utilization by opossum red cells averaged 3.5 mumol, at pH 8.1, 9.5 mumol/ml cells/hr were utilized. Red cells suspended in leukocyte-free plasma utilized D-glucose at a rate of 3.0 mumol/ml/hr at pH 7.5. Seven percent of D-glucose flowed through the pentose phosphate pathway; this rate increased 11-fold by methylene blue stimulation. The amount of D-glucose recycled through the pentose phosphate pathway increased 300-fold in the presence of the redox dye.     

The combination of high glucose and advanced glycation end-products (AGEs) inhibits the mineralization of osteoblastic MC3T3-E1 cells through glucose-induced increase in the receptor for AGEs.

Type 1 diabetes mellitus is known to be associated with reduced bone mass and increased bone fractures. This is thought to be due to a decrease in osteoblastic bone formation rather than an increase in osteoclastic bone resorption, but the precise mechanism is unknown. In this study, we examined whether or not high glucose or advanced glycation end-products (AGEs), which play key roles in the pathogenesis and complications of diabetes, affect the differentiation of osteoblastic MC3T3-E1 cells. First, MC3T3-E1 cells were incubated in media containing either 22 mM glucose, 22 mM mannitol, 300 microg/ml AGE2, or 300 microg/ml AGE3. Each of these agents alone did not affect the mineralization of the cells by von Kossa staining and Alizarin red staining. However, high glucose but not mannitol or AGEs markedly increased mRNA expression of AGE receptor (RAGE) by real-time PCR. Next, we examined the combined effects of high glucose and AGEs on the differentiation of MC3T3-E1 cells. The combination of 22 mM glucose and 300 microg/ml AGE2 significantly inhibited the mineralization of MC3T3-E1 cells, and 22 mM glucose in combination with either 300 microg/ml AGE2 or AGE3 apparently decreased osteocalcin mRNA expression. These results suggest that high glucose or AGEs alone might have no effect on osteoblastic differentiation, but their combination could additionally or synergistically inhibit osteoblastic mineralization through glucose-induced increase in RAGE expression.