Diagnostic tools for rapid detection and quantification of Weissella ceti NC36 infections in rainbow trout.

Weissellosis of rainbow trout is caused by the Gram-positive bacteria Weissella ceti and has been reported in China, Brazil and the United States. This disease can result in high mortality in market-sized fish and thus can cause significant economic loss. Thus far, phenotypic characterization and 16S rRNA sequencing have been used to confirm a Weissellosis diagnosis. Here, we present the development of PCR-based diagnostic tools for the rapid identification and quantification of W. ceti within bacteriological culture and infected tissues. A duplex PCR, which amplifies both genus- and strain-specific targets, positively identifies isolates as W. ceti NC36. A qPCR assay was also developed to quantify pathogen load from infected tissues, using a W. ceti NC36 unique locus. A proof of concept study was performed to demonstrate that quantification using traditional plate count methods and qPCR were significantly correlated when assessed from infected brain and spleen tissue. These tools were also used to confirm diagnosis of Weissellosis in a commercial rainbow trout farm during an outbreak investigation. These are the first diagnostic tools developed for identification and quantification of W. ceti infection within rainbow trout, contributing to rapid Weissellosis diagnosis, enhanced pathogen surveillance and epidemiological studies.

Effect of thyrotoxicosis on gluconeogenesis from alanine in the perfused rat liver.

The influence and mechanism of action of T4 treatment on hepatic gluconeogenesis from alanine was studied in isolated rat livers. Conversion of alanine into glucose was increased markedly in livers of thyrotoxic rats compared to that in normal rats. Estimation of metabolic intermediates of the gluconeogenic pathway showed that T4 treatment produced forward cross-over between pyruvate and phosphoenolpyruvate, which suggests that this point is a control site. Alanine transport into the liver cells was apparently active since the alanine level was higher within the hepatocytes than in the extracellular space. Furthermore, T4 treatment stimulated the transport of alanine into the liver cells. alpha-Aminoisobutyric acid uptake was significantly higher by liver of thyrotoxic animals than of normal rats. The hepatic level of cAMP was also higher in experimental livers than control livers. These studies suggest that T4 stimulates hepatic gluconeogenesis from alanine and the underlying mechanism involves an increase in both the transport of amino acid into the liver cell and the conversion pyruvate to phosphoenolpyruvate.

Effects of ethanol on glucose turnover in pregnant rats.

Glucose turnover was measured in term pregnant rats fed ethanol (30% of caloric intake) throughout gestation. Ethanol ingestion significantly reduced maternal weight gain and term fetal body weight when compared to pair-fed or ad libitum-fed controls. At term the blood glucose level and 6-3H-glucose turnover were reduced when compared to either control group. The rate of gluconeogenic recycling, indicated by the difference between 6-3H and 6-14C-glucose turnover determinations, was reduced by ethanol ingestion to half that of the control groups. Glucose turnover correlated with both conceptus weight and blood glucose level. Impaired maternal glucose homeostasis, including a reduced gluconeogenic response to the metabolic demands of late pregnancy, may thus contribute to the effects of ethanol on intrauterine growth.

Ethanol and glucose-deprivation neurotoxicity in cortical cell cultures.

The toxic effects of ethanol on rat cortical cell cultures were compared with neuronal damage induced by glucose deprivation. Exposure to decreased glucose concentrations produced dose-dependent neuronal injury, as indicated by the release of lactate dehydrogenase (LDH) into the culture medium. Complete glucose deprivation resulted in mean LDH release that was more than 60% greater than that from sister cultures incubated in the presence of 5.5 mmol/L glucose. Exposure to ethanol (25, 50, or 100 mmol/L) similarly resulted in dose-related LDH release. The degree of injury resulting from complete glucose deprivation or 100 mmol/L ethanol approximated that produced by exposure to 100 mmol/L glutamic acid. Ethanol did not significantly alter LDH release from cultures consisting of only astrocytes. Both effects were inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV). Glutamate levels were increased in the culture medium to 191% +/- 8% of the control value after glucose deprivation (P < .001) and to 186% +/- 16% after exposure to 100 mmol/L ethanol (P < .01). 3H-glutamate uptake by cultured astrocytes was reduced by glucose deprivation and by ethanol. This range of ethanol concentrations has previously been shown to inhibit hexose uptake by cultured astrocytes. The present results suggest that decreased glucose uptake by astrocytes in the presence of ethanol impairs their uptake of glutamate, which contributes to excitotoxic neuronal injury.

Effects of ethanol ingestion on glucose transporter-1 protein and mRNA levels in rat brain.

In the normal adult brain, glucose provides 90% of the energy requirement, as well as substrate for nucleic acid and lipid synthesis. We have previously observed that ethanol impairs hexose uptake by rat astrocytes in culture. In the present study, male Sprague-Dawley rats, 200-250 g, were fed liquid diet in which 36% of the calories were derived from ethanol (EF) for 4 weeks. Controls were fed ad libitum (AF) or pair-fed (PF) an equicaloric diet without ethanol. Blood glucose levels did not differ between the groups at the time of study. Glucose transport by brain plasma membranes was characterized by cytochalasin B binding and showed a slight increase in transporter number (mean +/- SEM of 4 experiments = 76.4 +/- 2.5 pmoles/mg protein in EF vs. 69.5 +/- 1.0 in PF) with no change in affinity (1.8 +/- 0.1 nM-1 in EF and 1.6 +/- 0.1 in PF). Glucose transporter, GLUT-1, was increased on Western blots. In contrast, Northern analysis of cortical tissue, using a rat brain glucose transporter cDNA insert (1.59 kb Bgl II fragment of pSPGT-1), showed a 23 to 35% decrease in steady-state levels of glucose transporter mRNA. GLUT-1 mRNA, localized in brain sections by in situ hybridization histochemistry, showed marked reductions in choroid plexus and hippocampus following ethanol treatment. Ethanol appears to have multiple effects on brain GLUT-1.

Decreased glucose transporter 1 gene expression and glucose uptake in fetal brain exposed to ethanol.

Using pregnant rats fed equicaloric liquid diets (AF, and libitum-fed controls; PF, pair-fed controls; EF, ethanol-fed), we have previously shown that maternal alcoholism produces a specific and significant decrease of glucose in the fetal brain, which is accompanied by growth retardation. To further define the mechanisms of ethanol-induced perturbations in fetal fuel supply, we have examined (i) the uptake of 2-deoxyglucose (2-DG) by dissociated brain cells from fetal rats that were exposed to ethanol in utero and (ii) the steady-state levels of the glucose transporter-1 (GT-1) mRNA. A 9% decrease in brain weight (P less than 0.001) and a 54.8% reduction in 2-DG uptake into brain cells (P less than 0.02) were found in offspring of EF mothers compared to the AF group. Brain weight correlated with the rate of 2-DG uptake (P less than 0.05). Northern blot analysis showed a 50% reduction of GT-1 mRNA in EF brain relative to that in the AF and PF groups. We conclude that glucose transport into the brain is an important parameter altered by maternal ethanol ingestion.

AUUUA-specific mRNA binding proteins in astrocytes.

Binding of ribonucleoproteins to specific regions of mRNA can alter mRNA stability. This level of posttranscriptional regulation has been shown to play a major role in gene expression of eukaryotic cells. This process involves the binding of ribonucleoproteins to specific region(s) of unstable, rapidly degrading mRNAs such as those found in various cytokines, lymphokines, and oncogenes, thereby increasing the mRNA's stability. In many instances the instability of the mRNA has been mapped to an AU-rich motif in the 3' untranslated region. We transcribed RNA molecules containing four reiterations of an AUUUA motif, and demonstrated with RNA- band shift experiments that the AUUUA motif complexes with phosphorylated AUUUA-specific 43-47 kDa mRNA binding protein(s) found in the cytosol of both rat brain and cultured rat astrocytes.

Maternal alcohol ingestion inhibits fetal glucose uptake and growth.

The distribution of maternally-derived glucose was determined in selected tissues of fetuses from ethanol-fed (EF) rats and from pair-fed (PF) and ad lib-fed (AF) controls. Maternal ethanol ingestion resulted in reduced fetal brain and liver weights and lower liver and lung glycogen levels compared to those of the PF or AF control groups. In addition, experimental fetuses exhibited reduced uptake of maternally-derived [3H] 2-deoxy-D-glucose (2-DG) by placenta and fetal brain. Fetal body, liver, lung, and brain weights correlated with fetal plasma 3H activity and with the fetal:maternal plasma 3H ratio, an indicator of the rate of placental glucose transfer. Brain weight correlated with 2-DG content per gram tissue weight. These observations suggest that reduced nutrient availability due to impaired placental transfer plays a role in the intrauterine growth retardation associated with maternal ethanol ingestion.

Growth patterns of the offspring of alcohol-fed rats.

The role of altered glucose metabolism in the growth retardation associated with the Fetal Alcohol Syndrome has been difficult to assess because previously used experimental procedures have frequently substituted ethanol for dietary carbohydrate. Consequently, there is a severe reduction of the carbohydrate content of the diet. This study examined brain and liver development at 7, 15, 21, and 42 days of age in the male offspring of ethanol-fed rats which received a liquid diet in which carbohydrate and protein content were equal to that of the control diet. Ethanol provided 30% of the daily calories and was substituted isocalorically for fats. Body and liver weights of the offspring of these ethanol-fed rats were significantly lower than controls at all ages studied, and the mean brain weight was decreased at 7 and 15 days of age but not subsequently. Brain protein content was significantly lower in the experimental group at 7 and 15 days of age, and liver protein content was reduced at 15, 21, and 42 days of age. The DNA content was not affected in either organ by exposure to ethanol in utero. Increasingly carbohydrate and total caloric intake moderates, but does not eliminate, the effects of maternal ethanol ingestion on offspring growth.

Interrelation between the effects of ethanol and thyroid hormones on gluconeogenesis from alanine in perfused rat liver.

Ethanol and thyroid hormones are among various factors that can alter hepatic gluconeogenesis, and the present study was done to determine the interrelation between the influence of ethanol and of T4 on glucose synthesis from alanine in isolated rat liver. With a sufficient supply of alanine, 20 mM ethanol enhanced glyconeogenesis nearly 60% in normal rat liver; higher ethanol concentrations, i.e., 40 or 80 mM, produced no further change in glucose synthesis. In contrast, 20, 40, or 80 mM ethanol had no effect on an already accelerated rate of gluconeogenesis in T4-treated rat liver. the redox state of the liver cytosol, as indicated by the L/P ratio and the alpha-GP/DHAP ratio, was markedly increased by ethanol in normal rat liver and, to a lesser extent, in thyrotoxic rat liver. Ethanol decreased pyruvate, PEP, and 3PGA but increased DHAP, F6P, and G6P concentrations in normal rat liver. In thyrotoxic rat liver per se, the pyruvate level was decreased whereas the levels of intermediates in the gluconeogenic sequence from OAA to 3PGA, and of alanine uptake and on pyruvate conversion to PEP. NADH produced by ethanol oxidation enhanced 3PGA conversion to DHAP in T4-treated as well as in normal rat liver. Despite these effects of ethanol on the gluconeogenic pathway, T4 stimulation of the rate of gluconeogenesis was not affected by ethanol. The results suggest that with an adequate supply of alanine as substrate, ethanol stimulates gluconeogenesis in normal rat liver but has no affect on an already enhanced gluconeogenesis in thyrotoxic rat liver.

Ethanol ingestion during pregnancy: effects on pregnant rats and their offspring.

A dose-response effect of ethanol intake on pregnant rats and their offspring has been studied. Ingestion of 36% ethanol diet reduced daily food consumption and attenuated gain in body weight relative to mothers fed control diet ad libitum. Mean values of survived litter size and body weight of offspring were significantly less than those of controls, although pair-fed controls ingested the same amount of food as ethanol-fed mothers. Offspring of 36% ethanol-fed animals failed to "catch up" in body weight relative to control pups. In contrast, feeding of 20% or 13% ethanol liquid diet did not adversely affect mothers and their offspring. Finally, administration of ethanol as 20% v/v drinking solution ad libitum resulted in decreased food intake as well as gain in body weight of pregnant rats and their offspring. The data suggest that ethanol ingestion in relatively smaller doses had no effect on maternal weight, litter size survival and growth of offspring, whereas relatively high ethanol doses adversely affected pregnant rats and their offspring.

Hepatic insulin binding following in utero exposure to ethanol.

Insulin binding to liver membranes has been studied in term fetuses of rats fed ethanol-containing liquid diet during pregnancy . Pair-fed and ad libitum-fed controls received liquid diet in which maltose-dextrins were substituted isocalorically for ethanol. Food consumption and body weigh gain of ethanol- imbibing dams were 35% and 70% less than their ad libitum counterparts respectively. Ethanol-fed rats also exhibited less gain in body weight than pair-fed controls despite isocalorically equivalent food intake. The number of live pups was not different among the various groups; however, liver weight of fetuses exposed to ethanol in utero was 47% less than those of the pups of ad libitum control dams and 28% less than those of the offspring of pair-fed control rats. Insulin binding to liver membranes of fetuses exposed to ethanol in utero was lower than that of ad libitum controls but was not significantly different from that of the pair-fed control animals. Average affinity profiles showed a reduction in K at all levels of receptor occupancy in the fetuses of ethanol-fed rats. For fetuses of the pair-fed group, K was reduced only at fractional occupancy below 20% but not at higher fractional occupancy. Because of the similarity of insulin binding in the fetuses of the ethanol-fed rats and their pair-fed counterparts, effects of ethanol on insulin binding cannot account for the reduced hepatic glycogen stores previously reported in term fetuses.

Ethanol inhibition of insulin secretion by perifused rat islets.

In vivo and in vitro effects of ethanol on the kinetics of insulin secretion in response to glucose and tolbutamide were studied in perifused rat islets. Phases I and II insulin response to 16.7 mM glucose was decreased 46% and 48%, respectively, in islets of rats given ethanol intragastrically 1 g/kg 1 h prior to sacrifice. Mean blood ethanol levels at the time of animal sacrifice were 19.4 mmol/l. The magnitude of insulin suppression was not significantly enhanced with higher ethanol doses, 2 or 3 g/kg, although mean blood ethanol levels increased to 25.9 and 60.3 mmol/l, respectively. Similarly, significant inhibition of both phases of insulin response to glucose occurred when ethanol 1 or 3 g/kg was given intraperitoneally instead of orally. Ethanol had no effect on insulin secretion when given orally 4 h instead of 1 h prior to islet isolation. Ethanol, 65 mmol/l, added directly to rat islets perifusate simultaneously with 16.7 mM glucose decreased both phases I and II insulin response nearly half; whereas addition of 21.7 instead of 65 mmol/l ethanol had no effect. Pre-treatment of islets with 21.7 or 65 mmol/l ethanol during 30 min basal islets perifusion period had no effect on subsequent insulin response to 16.7 mM glucose. Insulin response to 10 mM tolbutamide was decreased nearly 81% by the simultaneous presence of 65 mmol/l ethanol in islets perifusate.

Effects of ethanol on fetal fuels and brain growth in rats.

The mechanism of fetal brain growth retardation caused by maternal alcoholism is unclear. In this study we examined fuel concentrations in brain and blood samples and their relationship to brain growth in term fetuses of rats fed ethanol during pregnancy. The offspring of ethanol-fed (EF) rats showed a significant decrease in body and brain weights compared with those of pair-fed (PF) control rats and control rats given free access to food (ad libitum fed) (AF). The EF and PF rats consumed nearly 20% less food than the AF rats, and both groups showed a slight but significant reduction of the maternal blood glucose level. In PF rats, beta-hydroxybutyrate concentration was increased in maternal as well as in fetal blood and brain samples, but no adverse effect of this magnitude of maternal undernutrition was observed on fetal body or brain weights. In EF fetuses, plasma glucose and pyruvate levels were decreased and lactate levels were increased, resulting in a nearly twofold increase in the lactate-to-pyruvate ratio when compared with levels in control fetuses. The beta-hydroxybutyrate--to-acetoacetate ratio was increased because of low plasma acetoacetate concentration. In EF fetuses, brain glucose and pyruvate levels were decreased. Fetal brain weight showed a positive correlation with brain glucose concentration and a negative correlation with the brain concentrations of lactate or ketones. It is surmised that an aberrant fetal fuel mixture may play a role in the fetal growth retardation associated with maternal alcoholism.

Fetal alcohol syndrome: glucose and liver metabolism in term rat fetus and neonate.

UNLABELLED: The combined effects of chronic ethanol ingestion and fasting (24-hr fast, except water, prior to delivery) were examined in term pregnant rats and their offspring. Rats fed liquid diet containing 5% (w/v) ethanol (EF) consumed fewer calories than those fed control diet and exhibited reduced weight gain relative to either ad libitum-fed (AF) or pair-fed (PF) controls. While the number of live fetuses at term was unaffected, fetal body and liver weights were reduced in EF litters. Blood glucose levels were significantly lower in EF fetuses although maternal glucose levels did not differ between the groups. Liver glycogen levels also were reduced in EF fetuses, without any change in plasma immunoreactive insulin or immunoreactive glucagon levels. Both active and total glycogen synthase and phosphorylase were significantly lower in livers of EF fetuses than in livers of control fetuses. After delivery, blood glucose and plasma immunoreactive insulin levels fell more slowly in EF neonates than in either control group, but EF neonates remained hypoglycemic at 4 hr postnatal, whereas glycemia in both control groups had recovered to normal. Plasma immunoreactive glucagon levels in EF were elevated during the first 2 hr following delivery relative to either AF or PF controls, and hepatic glycogen levels were reduced in EF neonates during the entire interval studied. CONCLUSION: Fetal exposure to ethanol in utero and to a short maternal fasting prior to delivery results in fetal growth retardation, hypoglycemia, hypoinsulinemia, and liver glycogen depletion at term. Also, both glycogen synthase (active and total) and phosphorylase (active and total) were decreased as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ethanol on hexose uptake by cultured rat brain cells.

The effects of ethanol on hexose uptake by glial cells was investigated using primary cultures prepared from term rat fetuses. Specific 3H 2-deoxy-D-glucose (2DG) uptake was significantly reduced by a 4-hr exposure to ethanol at concentrations of 25, 50, and 100 mM, but not 200 or 300 mM. The inhibitory effect of 50 mM ethanol increased with the duration of exposure, with 2DG uptake inhibited by 36% after 18 hr. Astrocytes cultured from the brains of term fetuses of rats fed ethanol during pregnancy showed essentially the same 2DG uptake response to in vitro ethanol treatment. Kinetics of 2DG uptake showed a significant decrease of Vmax in the presence of ethanol. No interaction was found between ethanol and insulin, which stimulated 2DG uptake and protein content of the cultures. The data suggest that ethanol can modulate hexose uptake by astrocytes cultured from fetal rat brain. However, insulin actions on glial cells were not affected by ethanol.

Ethanol-induced intrauterine growth retardation: correlation with placental glucose transfer.

Placental transfer of glucose and alanine analogs was studied at term in ethanol-fed and control rats. Ethanol was provided as 30% of the caloric intake throughout gestation. Control groups received isocaloric liquid diet without ethanol by pair-feeding (PF) or ad libitum (AF). On the 22nd day of pregnancy, the rats were injected with a mixture of [3H]2-deoxyglucose and [14C]alpha-aminoisobutyric acid. The ratios of fetal:maternal plasma radioactivities 1 hr later were used to compare placental transfer between the groups. Mean +/- SE body weight of EF fetuses (4.54 +/- 0.07 g) was significantly lower than that of PF (4.88 +/- 0.06 g) or AF (5.17 +/- 0.09 g) fetuses. Maternal ethanol ingestion reduced placental transfer of 2-deoxyglucose and alpha-aminoisobutyric acid by 12% and 35%, respectively. Placental transfer of both analogs was not affected in the PF controls. The weight of EF fetuses correlated (p less than 0.001) with transfer of 2-deoxyglucose to the fetus. This relationship was also found in the control groups. Fetal body weight did not show a strong correlation with alpha-aminoisobutyric acid transfer. Thus, impaired transfer of glucose to the fetus may play a significant role in the growth retardation observed in fetuses of ethanol-fed rats.

Effects of ethanol on glucose utilization by cultured mammalian embryos.

We have previously observed correlations between placental glucose transfer and growth of fetuses of ethanol (EtOH)-fed and control rats. In the present study, whole mammalian embryos were used to define the interaction of glucose supply and the effects of EtOH on growth and differentiation. Rat embryos were cultured in 75% normal rat serum from day 9.5 to day 11.5 of gestation. EtOH produced dose-dependent reductions of embryo protein content (mean +/- SEM = 212 +/- 5, 171 +/- 11, 141 +/- 16, and 113 +/- 9 micrograms/embryo in the presence of 0, 25, 50, and 100 mM EtoH, respectively). Somite number was 25.7 +/- 0.3, 23.4 +/- 0.7, 21.8 +/- 0.7, and 21.1 +/- 0.4 under the same conditions. Exposure to ethanol during the first 24 hr in culture decreased embryo protein content to the same extent as exposure for the entire 48-hr culture period. After 46 hr in culture, control and ethanol-exposed embryos were incubated with 14C-glucose for 2 hr. Ethanol produced dose-dependent reductions of CO2 production, anabolic utilization, lactate release, and total glucose utilization. Glucose supplementation (300 mg/dl) significantly increased embryo protein content and each of these glucose utilization parameters. When glucose utilization was expressed relative to embryo protein content, incorporation of the label into embryonic tissues was significantly reduced by ethanol and increased by glucose supplementation. Embryo protein content correlated closely (r = 0.871, p less than 0.0001) with anabolic glucose utilization. Thus, ethanol directly affects embryo glucose utilization, both as an energy source and as a synthetic substrate, in addition to its effects on placental glucose transfer.

Effects of ethanol on DNA, RNA, and protein synthesis in rat astrocyte cultures.

Brain growth retardation is a major feature of the fetal alcohol syndrome (FAS). Insulin and insulin-like growth factors (IGF-I and IGF-II) exert significant growth-promoting effects on the central nervous system (CNS). The present study examined the effects of ethanol and its interactions with growth factors on the incorporation of labeled precursors into DNA, RNA, and protein in primary astrocyte cultures prepared from term fetal rats. Cultures were exposed to ethanol for 18hr in serum-free medium before measuring nucleoside or amino acid incorporation into acid-precipitable cell constituents. Under basal conditions, ethanol induced dose-dependent changes in the rates of incorporation of tritiated thymidine, uridine, and valine. The fraction of the total thymidine uptake that was incorporated into DNA was reduced in the presence of 100 and 200 mM ethanol. Effects on uridine and valine incorporation paralleled cell uptake. Insulin (10(-6) M) and IGF-I (10(-9) M) increased (p less than 0.01) incorporation of radiolabeled thymidine, uridine, and valine. Analysis of variance indicated highly significant interactions between ethanol and the effects of growth factors on incorporation of both nucleosides and valine. Interference with the action of neurotrophic factors may be a significant factor in fetal brain growth retardation associated with maternal ethanol ingestion.

Effects of ethanol on glucose transporter expression in cultured hippocampal neurons.

Glucose transport was studied in primary hippocampal neuron cultures exposed to ethanol. Immunofluorescent staining with antibodies against neuron-specific enolase and glial fibrillary acidic protein identified approximately 95% of the cultured cells as neurons. Western blot analysis was conducted with polyclonal antisera to glucose transporter isoforms GLUT1 and GLUT3. As previously seen in astrocytes, GLUT1 protein was regulated by the culture medium glucose content. Exposure to 50 and 100 mM of ethanol for 5 hr induced dose-dependent reductions in GLUT1 and GLUT3 protein. In contrast, GLUT1 mRNA abundance was increased relative to controls under the same conditions. Glucose uptake, measured with the nonmetabolized analog, 2-deoxy-D-glucose, was reduced by 50 and 100 mM of ethanol in four experiments. These results indicate a direct effect of ethanol on neuronal glucose transporter expression, which may play a role in the neurotoxic effects of alcohol.