Maternal dexamethasone administration and the maturation of perirenal adipose tissue of the neonatal sheep.

Maternal dexamethasone administration promotes fetal maturation such that thermoregulation is improved following premature delivery and is thus comparable with a full term birth. In the present study we determined the impact of dexamethasone on both the mothers' metabolic status together with adipose tissue function in the newborn. Glucocorticoid action, adipokine gene expression and mitochondrial protein abundance were measured in perirenal adipose tissue of neonatal sheep that were born into either a warm (30 degrees C) or cool (15 degrees C) ambient temperature at 140 days of gestation (dGA; term approximately 147 dGA), either two days after maternal dexamethasone administration, or at 146 dGA for controls. Dexamethasone administration resulted in a reduction in maternal food intake in conjunction with raised plasma cortisol and free triiodothyronine. In offspring of dexamethasone administered mothers, plasma cortisol was lower and non-esterified fatty acids (NEFA) higher than controls. Glucocorticoid receptor (GR), 11beta-hydroxysteroid dehydrogenase (11beta-HSD1), interleukin-6 and uncoupling protein (UCP)1 and 2 mRNA together with voltage dependent anion channel, cytochrome c protein and UCP1 abundance were all increased by dexamethasone administration and being born into a cool ambient temperature. Gene expression of tumor necrosis factor alpha, adiponectin and peroxisome proliferator-activated receptor transcription factor gamma were unaffected by dexamethasone. The abundance of mRNA for the GR, 11beta-HSD1, UCP1 and 2 mRNA together with each protein were positively correlated to plasma NEFA and negatively correlated to plasma cortisol. In conclusion, despite reduced maternal food intake dexamethasone promotes maturation of glucocorticoid action and mitochondrial protein abundance in the newborn, an adaptation dependent on delivery temperature.

Nutritional manipulation between early to mid-gestation: effects on uncoupling protein-2, glucocorticoid sensitivity, IGF-I receptor and cell proliferation but not apoptosis in the ovine placenta.

In sheep, modest maternal nutrient restriction (NR) over the period of rapid placental growth restricts placentome growth and results in offspring in which glucocorticoid action is enhanced. Therefore, this study investigated the placental effects of early to mid-gestational NR on glucocorticoid receptor (GR), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), uncoupling protein-2 (UCP2), and IGF type-I receptor (IGF-IR) mRNA abundance together with cell proliferation and apoptosis as determined histologically, and the mitochondrial proteins voltage-dependent anion channel and cytochrome c that are involved in apoptosis. Placenta was sampled at 80 and 140 days gestation (dGA; term ~147 dGA). NR was imposed between 28 and 80 days gestation when control and nutrient-restricted groups consumed 150 or 60% respectively of their total metabolizable energy requirements. All mothers were then fed to requirements up to term. Total fetal placentome weights were decreased by NR at 80 dGA but were heavier at 140 dGA following 60 days of nutritional rehabilitation. GR and UCP2 mRNA abundance increased whilst 11betaHSD2 mRNA decreased with gestational age. NR persistently up-regulated GR and UCP2 mRNA abundance. 11betaHSD2 mRNA was reduced by NR at 80 dGA but increased near to term. IGF-IRmRNA abundance was only decreased at 80 dGA. Placental apoptosis and mitochondrial protein abundance were unaffected by NR, whereas cell proliferation was markedly reduced. In conclusion, placental UCP2 and local glucocorticoid action are affected by the gestational nutritional status and may result in the offspring showing enhanced glucocorticoid sensitivity, thereby predisposing them to disease in later life.

Maternal nutrient restriction in early pregnancy programs hepatic mRNA expression of growth-related genes and liver size in adult male sheep.

The liver is a major metabolic and endocrine organ of critical importance in the regulation of growth and metabolism. Its function is determined by a complex interaction of nutritionally regulated counter-regulatory hormones. The extent to which hepatic endocrine sensitivity can be programed in utero and whether the resultant adaptations persist into adulthood is unknown and was therefore the subject of this study. Young adult male sheep born to mothers that were fed either a control diet (i.e.100% of total live weight-maintenance requirements) throughout gestation or 50% of that intake (i.e. nutrient restricted (NR)) from 0 to 95 days gestation and thereafter 100% of requirements (taking into account increasing fetal mass) were entered into the study. All mothers gave birth normally at term, the singleton offspring were weaned at 16 weeks, and then reared at pasture until 3 years of age when their livers were sampled. NR offspring were of similar birth and body weights at 3 years of age when they had disproportionately smaller livers than controls. The abundance of mRNA for GH, prolactin, and IGF-II receptors, plus hepatocyte growth factor and suppressor of cytokine signaling-3 were all lower in livers of NR offspring. In contrast, the abundance of the mitochondrial protein voltage-dependent anion channel and the pro-apoptotic factor Bax were up regulated relative to controls. In conclusion, maternal nutrient restriction in early gestation results in adult offspring with smaller livers. This may be mediated by alterations in both hepatic mitogenic and apoptotic factors.

Maternal nutritional programming of fetal adipose tissue development: differential effects on messenger ribonucleic acid abundance for uncoupling proteins and peroxisome proliferator-activated and prolactin receptors.

Maternal nutrient restriction at specific stages of gestation has differential effects on fetal development such that the offspring are programmed to be at increased risk of a range of adult diseases, including obesity. We investigated the effect of maternal nutritional manipulation through gestation on fetal adipose tissue deposition in conjunction with mRNA abundance for uncoupling protein (UCP)1 and 2, peroxisome proliferator-activated receptors (PPAR)alpha and gamma, together with long and short forms of the prolactin receptor (PRLR). Singleton-bearing ewes were either nutrient restricted (3.2-3.8 MJ day(-1) metabolizable energy) or fed to appetite (8.7-9.9 MJ day(-1)) over the period of maximal placental growth, i.e. between 28 and 80 d gestation. After 80 d gestation, ewes were either fed to calculated requirements, (6.7-7.5 MJ day(-1)), or to appetite (8.0-10.9 MJ day(-1)). At term, offspring of nutrient-restricted ewes possessed more adipose tissue, an adaptation that was greatest in those born to mothers that fed to requirements in late gestation. This was accompanied by an increased mRNA abundance for UCP2 and PPARalpha, an adaptation not seen in mothers re-fed to appetite. Maternal nutrition had no effect on mRNA abundance for UCP1, PPARgamma, or PRLR. Irrespective of maternal nutrition, mRNA abundance for UCP1 was positively correlated with PPARgamma and the long and short forms of PRLR, indicating that these factors may act together to ensure that UCP1 abundance is maximized in the newborn. In conclusion, we have shown, for the first time, differential effects of maternal nutrition on key regulatory components of fetal fat metabolism.

Maternal endocrine adaptation throughout pregnancy to nutritional manipulation: consequences for maternal plasma leptin and cortisol and the programming of fetal adipose tissue development.

Maternal nutrient restriction at specific stages of gestation has differential effects on fetal development such that the offspring are programmed to be at increased risk of adult disease. We investigated the effect of gestational age and maternal nutrition on the maternal plasma concentration of leptin and cortisol together with effects on fetal adipose tissue deposition plus leptin, IGF-I, IGF-II ligand, and receptor mRNA abundance near to term. Singleton bearing ewes were either nutrient restricted (NR; consuming 3.2-3.8 MJ/d of metabolizable energy) or fed to appetite (consuming 8.7-9.9 MJ/d) over the period of maximal placental growth, i.e. between 28 and 80 d gestation. After 80 d gestation, ewes were either fed to calculated requirements, consuming 6.7-7.5 MJ/d, or were fed to appetite and consumed 8.0-10.9 MJ/d. Pregnancy resulted in a rise in plasma leptin concentration by 28 d gestation, which continued up to 80 d gestation when fed to appetite but not with nutrient restriction. Plasma cortisol was also lower in NR ewes up to 80 d gestation, a difference no longer apparent when food intake was increased. At term, irrespective of maternal nutrition in late gestation, fetuses sampled from ewes NR in early gestation possessed more adipose tissue, whereas when ewes were fed to appetite throughout gestation, fetal adipose tissue deposition and leptin mRNA abundance were both reduced. These changes may result in the offspring of NR mothers being at increased risk of obesity in later life.

Maternal nutrient restriction during placental growth, programming of fetal adiposity and juvenile blood pressure control.

Epidemiological and experimental studies have demonstrated that maternal undernutrition during pregnancy is associated with abnormal placental growth. In sheep, maternal nutrient restriction over the period of rapid placental growth (30-80 days) restricts placentome growth. Then following adequate nutrition up to term (147 days), placental mass is greater in association with a higher total abundance of the predominant placental glucose transporter-1. The resulting lambs are larger at birth, have heavier kidneys with an increased expression of the glucocorticoid-responsive type 1 angiotensin II receptor. Near to term, these fetuses possess more adipose tissue, the endocrine sensitivity of which is markedly enhanced. For example, the abundance of mRNA for 11beta-hydroxysteroid dehydrogenase type 1, which catalyses the conversion of cortisone to bio-active cortisol is increased. This is associated with a higher abundance of both leptin and glucocorticoid receptor mRNA. At 6 months of age, the juvenile offspring of nutrient restricted ewes have lower resting blood pressure that was positively correlated with plasma cortisol concentration, suggesting their blood pressure could be more strongly driven by circulating cortisol. These offspring also exhibited a greater pressor response to vasoconstrictor challenges, but showed no difference in vasodilatory response. At this age, the kidney weight was similar between groups, but the abundance of cytochrome c in kidney mitochondria was enhanced in lambs born to nutrient restricted ewes that could indicate increased mitochondrial activity. Reduced maternal nutrition during the period of rapid placental growth may therefore contribute to hypertension in later life through physiological and vascular adaptations during fetal life.

Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis.

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.

Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.

Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

Salmonella pathogenicity island 2 influences both systemic salmonellosis and Salmonella-induced enteritis in calves.

We have used signature-tagged mutagenesis to identify mutants of the host-specific Salmonella enterica serotype Dublin which were avirulent in calves and/or BALB/c mice. A mutant with a transposon insertion in the sseD gene of Salmonella pathogenicity island 2 (SPI-2), which encodes a putative secreted effector protein, was identified. This mutant was recovered from the bovine host but not from the murine host following infection with a pool of serotype Dublin mutants. However, a pure inoculum of the sseD mutant was subsequently shown to be attenuated in calves following infection either by the intravenous route or by the oral route. The sseD mutant was fully invasive for bovine intestinal mucosa but was subsequently unable to proliferate to the same numbers as the parental strain in vivo. Both the sseD mutant and a second SPI-2 mutant, with a transposon insertion in the ssaT gene, induced significantly weaker secretory and inflammatory responses in bovine ligated ileal loops than did the parental strain. These results demonstrate that SPI-2 is required by serotype Dublin for the induction of both systemic and enteric salmonellosis in calves.

Effect of maternal nutrition on brown adipose tissue and its prolactin receptor status in the fetal lamb.

We investigated the influence of maternal nutritional enhancement during the second half of gestation on prolactin receptor (PRLR) abundance in fetal brown adipose tissue (BAT) and liver close to term (i.e. 141-144 d gestation). Ewes were provided with 100% (i.e. control; n = 8) or 150% (i.e. well-fed; n = 7) of their metabolic requirements from 80 to 144 d gestation. Crude plasma membranes were prepared from fetal BAT and hepatic tissue, and individual molecular weight isoforms for the long and short forms of the PRLR were detected by immunoblotting. Mitochondrial preparations were prepared from BAT to measure the amount of the BAT-specific mitochondrial uncoupling protein-1 and its thermogenic activity (i.e. guanosine 5'-diphosphate binding). Fetuses sampled from well-fed ewes were heavier (controls, 3927 +/- 196 g; well-fed, 4783 +/- 219 g; p = 0.01) but possessed less BAT per kilogram body weight (controls, 5.92 +/- 0.43 g/kg; well-fed, 3.85 +/- 0.19 g/kg; p = 0.001), which had a greater uncoupling protein-1 abundance (controls, 56 +/- 5% of reference; well-fed, 78 +/- 9% of reference; p < 0.01) and higher thermogenic activity (controls, 157 +/- 41 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; well-fed, 352 +/- 36 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; p < 0.01) than controls. Multiple isoforms of the long and short forms of the P1LR were detected in all tissues. BAT from well-fed fetuses had a higher abundance of the 15-kD isoform of the long form of the PRLR (controls, 1.6 +/- 0.4 densitometric units; well-fed, 16.3 +/- 2.0 densitometric units; p < 0.001). This isoform was not detected in hepatic tissue. Maternal nutrient intake had no effect on any other isoforms of the PRLR in BAT or liver. In conclusion, increasing the quantity of feed provided in late gestation acts to promote fetal weight and BAT maturation, the combination of which will enhance neonatal viability.

Effect of maternal dexamethasone treatment and ambient temperature on prolactin receptor abundance in Brown adipose and hepatic tissue in the foetus and new-born lamb.

We investigated the influence of maternal dexamethasone treatment and ambient temperature on prolactin receptor (PRLR) abundance in brown adipose tissue (BAT) and hepatic tissue from foetuses and 6-h-old lambs delivered by caesarean section. Lambs were either delivered into a warm (30 degrees C; WD) or cool (15 degrees C; CD) ambient temperature at 140 days gestation, 2 days after dexamethasone treatment, or at 146 days gestation for controls. Uncoupling protein-1 (UCP1) content of BAT was higher in dexamethasone-treated groups compared to controls. A range of tissue-specific PRLR isoforms was detected. For the long form of PRLR in BAT these isoforms had molecular weights of 66, 54, 34 and 19 kD compared with 88, 76, 66, 58, 54 and 48 kD in liver. In BAT, isoforms of the short form of PRLR had molecular weights of 66, 62, 54, 48, 33 and 31 kD compared with 82, 66, 56, 54, 48, 40 and 33 kD in liver. Dexamethasone treatment in CD lambs resulted in higher abundance of the 54 kD isoform of the short form of PRLR in liver, whilst in BAT dexamethasone resulted in a greater abundance of the 48 kD isoform of the short form, and lower abundance of the 66 kD isoform of the long form of PRLR, compared to controls. A negative correlation (r2 = 0.52) was observed between abundance of 66 kD isoform for the long form of PRLR and UCP1, compared with positive correlations (r2 = 0.58-0.60) for the abundance of the 54 and 48 kD isoforms for the short form of PRLR and UCP1. In conclusion, maternal dexamethasone treatment 1 week before term alters the abundance of PRLR isoforms in a tissue-specific manner. This response is dependent on ambient temperature after birth and may provide a critical endocrine signal for maximising non-shivering thermogenesis.

Nonselective effects of the putative phospholipase C inhibitor, U73122, on adenosine A1 receptor-mediated signal transduction events in Chinese hamster ovary cells.

Adenosine A1 receptors can signal, through Gi/o proteins, to inhibit adenylyl cyclase activity and also to stimulate phosphoinositide hydrolysis and the subsequent release of intracellular Ca2+ stores. The aminosteroid U73122 (1-[6-1[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione) has been widely used as an inhibitor of phospholipase C, the enzyme mediating phosphoinositide hydrolysis. Using U73122, we sought to selectively block signalling through the phospholipase C pathway, in Chinese hamster ovary (CHO-K1) cells heterologously expressing human adenosine A1 receptors. U73122 inhibited A1 receptor-mediated phosphoinositide hydrolysis, as measured by total inositol phosphate accumulation, over the concentration range 1-15 microM. However, over the same concentration range, it also appeared to inhibit A1 receptor-mediated inhibition of forskolin-stimulated cyclic AMP accumulation, A1 receptor agonist-promoted [35S]GTP-gammaS binding, and at the higher concentrations (10-15 microM) produced marked morphological changes, leading to cytolysis. The structural analogue of U73122, U73343 (1-[6-[[17beta-3-methoxyestra-1,3,5(10-trien-17-yl]amino]hexyl]-2, 5-pyrrolidone-dione), typically used as an inactive control compound, had little effect on these events. The data suggest that U73122 is not a selective inhibitor of phospholipase C activity, interfering with adenosine A1 receptor signalling generally, either at the pre-effector level involving Gi/o proteins, or as a consequence of the morphological changes it induces.