Substitution of starch for palm oil during gestation: impact on offspring survival and hepatic gene expression in the pig.

Piglet neonatal mortality rates are high (~20%), so nutritional strategies to reduce this are highly desirable. Maternal fat substitution (FS) may promote the preweaning survival of piglets by improving their energy status. Therefore, the aim of the present study was to investigate the effects of FS throughout pregnancy on offspring viability, together with the gene expression of stress-related markers in the liver. Sixteen pregnant sows were randomly allocated to one of two isocaloric diets, control (C) or FS in the form of palm oil, fed from 0 to 110 days gestation. Glucose tolerance was examined on Day 108. Median and low birthweight offspring were allocated to tissue sampling at either 7 days or 6 months postnatal age. In response to a glucose tolerance test, FS sows exhibited a raised glucose area under the curve with no change in basal glucose. Average piglet mortality (up to Day 28) was increased fourfold in the FS group, with surviving median-sized piglets exhibiting significantly lower fatty acid binding protein 1 (FABP1) expression at 7 days. There were no effects on the abundance of any other stress- or metabolic-related genes examined. Thus, this study demonstrates that maternal FS throughout gestation causes maternal glucose intolerance that may be linked to the observed increase in piglet mortality. However, the surviving offspring do not exhibit any detectable differences in postnatal growth or hepatic gene profile in later life.

Risk of infection and sepsis in severely injured patients related to single nucleotide polymorphisms in the lectin pathway.

BACKGROUND: Infectious complications remain a serious threat to patients with multiple trauma. Susceptibility and response to infection is, in part, heritable. The lectin pathway plays a major role in innate immunity. The aim of this study was to assess whether single nucleotide polymorphisms (SNPs) in three key genes within the lectin pathway affect susceptibility to infectious complications in severely injured patients. METHODS: A prospective cohort of severely injured patients admitted to a level I trauma centre between January 2008 and April 2011 were genotyped for SNPs in MBL2 (mannose-binding lectin 2), MASP2 (MBL-associated serine protease 2) and FCN2 (ficolin 2). Association of genotype with prevalence of positive culture findings and infection was tested by χ(2) and logistic regression analysis. RESULTS: A total of 219 patients were included, of whom 112 (51·1 per cent) developed a positive culture from sputum, wounds, blood or urine. A systemic inflammatory response syndrome (SIRS) developed in 139 patients (63·5 per cent), sepsis in 79 (36·1 per cent) and septic shock in 37 (16·9 per cent). Patients with a MBL2 exon 1 variant allele were more prone to positive wound cultures (odds ratio (OR) 2·51, 95 per cent confidence interval 1·12 to 5·62; P = 0·025). A MASP2 Y371D DD genotype predisposed to SIRS (OR 4·78, 1·06 to 21·59; P = 0·042) and septic shock (OR 2·53, 1·12 to 4·33; P = 0·003). A FCN2 A258S AS genotype predisposed to positive wound cultures (OR 3·37, 1·45 to 7·85; P = 0·005) and septic shock (OR 2·18, 1·30 to 4·78; P = 0·011). CONCLUSION: Severely injured patients with SNPs in MBL2, MASP2 Y371D and FCN2 A258S of the lectin pathway of complement activation are significantly more susceptible to positive culture findings, and to infectious complications, SIRS and septic shock than patients with a wildtype genotype.

Construction of a doxycycline inducible adipogenic lentiviral expression system.

To provide a tool for research on regulating adipocyte differentiation, tetracycline inducible (Tet on) lentiviral expression vectors under the control of an adipose-specific promoter were constructed. The lowest basal expression in the absence of doxycycline and most efficient dose-dependent, doxycycline-induced transient overexpression was observed using vectors constructed with a combination of Tetracycline Responsive Element (TRE) and reverse tetracycline-controlled TransActivator advanced (rtTAadv), transfected in white (3T3-L1) and brown (HIB-1B) preadipocytes cell lines. The results demonstrate that doxycycline adipogenic inducible expression can be achieved using a pLenti TRE / rtTA adv under the control of the truncated aP2 promoter in HIB-1B preadipocytes.

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue.

AIMS/HYPOTHESIS: Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues. METHODS: Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding. RESULTS: We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter-reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment. CONCLUSIONS/INTERPRETATION: The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails.

Gene expression of inflammatory markers in adipose tissue between obese women with polycystic ovary and normal obese women.

OBJECTIVE: The pathogenesis of polycystic ovary syndrome (PCOS), a common endocrine disease and metabolic disturbance, is still unknown. The aim of the study was to investigate whether patients with PCOS display increased expression of inflammatory markers in adipose tissue. PATIENTS AND METHODS: Two groups of women were investigated, those diagnosed with PCOS (n = 8) and age and BMI-matched normal women (n = 12). Their age was between 20-45 years and all subjects were apparently healthy and did not take any medications. Adipose tissue levels of mRNA of inflammatory markers were determined by use of real-time PCR. RESULTS: There were no differences between obese patients and obese PCOS in levels of adipocytokines. CONCLUSIONS: There were no effects of PCOS on the expression of any of the adipocytokines genes measured in subcutaneous adipose tissue.

Substrate specificity of the mammary tissue anionic amino acid carrier operating in the cotransport and exchange modes.

The substrate specificity of the rat mammary tissue high affinity, Na+-dependent anionic amino acid transport system has been investigated using explants and the perfused mammary gland. D-Aspartate appears to be transported via the high affinity, Na+-dependent L-glutamate carrier. Thus, D-aspartate transport by rat mammary tissue was Na+-dependent and saturable with respect to extracellular D-aspartate with a Km and Vmax of 32.4 microM and 49.0 nmol/2 min per g of cells respectively. The uptake of D-aspartate by mammary explants was cis-inhibited by L-glutamate and L-aspartate, but not by D-glutamate. L-glutamate uptake by mammary tissue explants was cis-inhibited by beta-glutamate, L-cysteate, L-cysteine sulfinate and dihydrokainate but not by DL-alpha-aminoadipate. In addition, dihydrokainate, but not DL-alpha-aminoadipate inhibited D-aspartate and L-glutamate uptake by the perfused gland. D-Aspartate efflux from mammary tissue explants was trans-accelerated by external L-glutamate in a dose-dependent fashion (50-500 microM). The effect of L-glutamate on D-aspartate efflux was dependent on the presence of extracellular Na+. D-Aspartate, L-aspartate and L-cysteine sulfinate (at 500 microM) also markedly trans-stimulated D-aspartate efflux from mammary tissue explants. In contrast, L-cysteine. D-glutamate, L-leucine, dihydrokainate and DL-alpha-aminoadipate were either weak stimulators of D-aspartate efflux or were without effect. D-Aspartate efflux from the perfused mammary gland was trans-stimulated by L-glutamate but not by D-glutamate and only weakly by L-cysteine (all at 500 microM). It appears that the mammary tissue high affinity anionic amino acid carrier can operate in the exchange mode with a similar substrate specificity to that of the co-transport mode.

The mechanism of L-glutamate transport by lactating rat mammary tissue.

The transport of L-glutamate by lactating rat mammary gland has been examined using both tissue explants and a perfused mammary preparation. L-Glutamate uptake by mammary tissue explants was predominantly via a Na(+)-dependent pathway: Li+, choline+ and NMDG+ could not substitute for Na+. L-Glutamate efflux from preloaded explants was also influenced by the transmembrane Na(+)-gradient. These results are consistent with (Na(+)-glutamate) cotransport. The Na(+)-dependent system for L-glutamate transport in tissue explants was saturable (Km = 112.5 +/- 19.7 microM; Vmax = 71.3 +/- 10.4 nmol/min per g cells) and selective for anionic amino acids. Thus, D- and L-aspartate were high affinity inhibitors of L-glutamate uptake whereas neutral amino acids were relatively ineffective. D-Aspartate inhibited L-glutamate uptake in a competitive fashion. L-Glutamate uptake by the perfused mammary gland was (a) Na(+)-dependent (b) saturable (Km = 18.1 +/- 4.9 microM; Vmax = 40.3 +/- 3.7 nmol/min per g tissue) and (c) selective for anionic amino acids. The results suggest that the (Na(+)-glutamate) cotransporter is situated in the blood-facing aspect of the mammary epithelium.

The effect of dietary polyunsaturated fatty acids (PUFA) on infection with the nematodes Ostertagia ostertagi and Cooperia oncophora in calves.

Diet-induced changes in the polyunsaturated fatty acid (PUFA) content of immune cells influences the immune phenotype that develops following infection. The aim of this study was to examine the effect of manipulating dietary PUFA supply on tissue fatty acids composition and immunity to a mixed infection with an abomasal and an intestinal nematode parasite in calves. Calves (n=24) were allocated into two treatment groups and fed 25 g/day of either fish oil (n-3 group) or a binary mixture of palm/rapeseed oil (normal group) as a supplement in milk replacer. Within each treatment group eight calves were infected with 2000 L3 Ostertagia ostertagi and Cooperia oncophora, three times per week for 8 weeks, the remaining calves were pair-fed uninfected controls. Faecal egg counts (FEC) were carried out twice weekly. At slaughter, the whole gut was removed intact for worm counts and tissue samples were taken for fatty acid analysis. Samples of abomasum, duodenum and mid-gut were also collected for immunohistological analysis. FEC were not significantly influenced by oil supplement but tended to remain higher in the palm/rapeseed oil-fed group (normal infected). The number of intestinal immature worms was significantly (p<0.05) higher in the n-3 group. Mucosal mast cell (MMC) and eosinophil numbers were significantly increased (p<0.05) by infection and were significantly lower (p<0.05) in the intestinal tissue of the fish oil supplemented and infected group (n-3 infected group). These results suggest that feeding an n-3 PUFA-rich supplement (fish oil) can influence cellular mediators of immunity to nematode infection. This is the first report of the establishment of patency and the subsequent development of immunity to a mixed infection with O. ostertagi and C. oncophora in calves undergoing early rumen development. The trend in the FEC, MMC and eosinophil numbers in the n-3 group suggests that decreasing the dietary n-6/n-3 PUFA ratio may be a worthwhile immunonutritional strategy for potentiating the immune response to nematode parasite infection in the calf.

Effect of dietary protein intake and intravenous glucose infusion on plasma concentrations of insulin-like growth factor-I in lambs.

Two separate experiments were carried out to examine the effect of dietary protein intake on basal and GH-stimulated plasma insulin-like growth factor-I (IGF-I) concentrations during either saline or glucose infusion into the jugular vein. In experiment 1, six castrated male lambs (27.1 +/- 1.2 kg live weight (LW)) were fed a diet restricted in both metabolizable energy (ME; 0.18 MJ/kg LW per day) and nitrogen (2.0 g/kg LW per day) intakes, while in experiment 2 a further six lambs were fed a similar restricted ME intake but an increased nitrogen intake (3.0 g/kg LW per day). In both experiments glucose (experiment 1, 0.009 mmol/kg LW per min; experiment 2, 0.015 mmol/kg LW per min) or saline (0.25 ml/min) was infused for 6 days and plasma samples were obtained from the jugular vein at hourly intervals on day 4 (basal) or on days 5 and 6 after an i.v. GH challenge. In experiment 1 there was no increase in plasma IGF-I concentrations in response to the GH challenge during saline infusion, but during glucose infusion the plasma concentration of IGF-I increased to a peak after 24 h and declined over the next 20 h. Basal concentrations of IGF-I, insulin and glucose were significantly higher during glucose infusion. In experiment 2 the area under the IGF-I peak in response to the GH challenge was the same for the infusions of saline and glucose but the peak value for IGF-I was significantly higher during glucose infusion due to higher concentrations in the basal period.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutrient-hormone interaction in the ovine liver: methionine supply selectively modulates growth hormone-induced IGF-I gene expression.

This study tested the hypothesis that specific amino acids are responsible for modulating the insulin-like growth factor-I (IGF-I) response to growth hormone (GH) in ovine hepatocytes. Cells were grown in media of defined amino acid composition, based on physiological concentrations (P.C.) of amino acids in sheep plasma. Relative to culture in 5 x P.C., amino acid limitation to 0.2 x P.C. had inhibitory effects on IGF-I RNA expression, peptide release and p70 S6 kinase phosphorylation (P<0.01 in each case). Limitation of methionine levels to 0.2 x P.C. against a background of 5 x P.C. for the other amino acids blocked GH-stimulated IGF-I peptide release and RNA expression, although basal expression was unaffected. In contrast, limitation of the other amino acids present in the culture medium had no effect on basal or GH-stimulated IGF-I expression. Selective methionine limitation to 0.2xP.C. levels had no effect on cellular or secretory protein synthesis rates relative to cells grown in complete 5 x P.C. medium but did cause a partial reduction in p70 S6 kinase phosphorylation, which was also observed when medium was selectively limited for other essential amino acids. The addition of rapamycin (5 ng/ml) to cells grown in 5xP.C. media completely abolished p70 S6 kinase phosphorylation (P<0.001), implicating mTOR in the response of S6 kinase phosphorylation to changing amino acid supply. By contrast, inclusion of rapamycin (100 ng/ml) had no effect on levels of IGF-I gene expression. These results indicate that methionine is the key limiting amino acid involved in the modulation of IGF-I expression in the ovine liver. This nutrient-hormone interaction is a highly selective phenomenon, occurring against a background of modest effects on general protein synthetic control. The partial inhibitory effects of methionine on mTOR activity are not sufficient to account for this selectivity of action.

Growth hormone and amino acid supply interact synergistically to control insulin-like growth factor-I production and gene expression in cultured ovine hepatocytes.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being regulated by GH, plasma IGF-I concentrations have been demonstrated to depend upon the level of dietary protein intake, with low protein diets being associated with reduced circulatory IGF-I levels. This inhibitory effect cannot be reversed by GH injection, suggesting that liver sensitivity to GH becomes impaired.To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media, containing various proportions (0.2, 1.0 and 5.0) of jugular amino acid concentrations in fed sheep. Production of IGF-I by these cells was measured after 24 and 48 h in culture by radioimmunoassay. In the first 24-h period basal IGF-I production was the same in all defined media, and GH caused an approximately 2-fold increase in IGF-I release in cells grown in 1.0xor 5.0xamino acid media (P<0.01). Although GH appeared to increase IGF-I release in this period for cells grown in 0.2xamino acid media, this effect was not statistically significant. In the period from 24-48 h in defined media, both basal and GH-stimulated IGF-I production was dependent on amino acid availability (P<0.05 and P<0.001 respectively). Factorial analysis of variance demonstrated a strong positive interaction (P<0.001) between the effects of amino acid availability and GH, such that GH increased IGF-I production by more than 2-fold in cells grown in 5.0xamino acid media (P<0.01) but had no effect on production by cells grown in 1.0xor 0.2xamino acid media. Measurement of steady state concentrations of exon 1-derived IGF-I mRNAs using an RNase protection assay demonstrated that the observed effects on IGF-I peptide secretion were strongly associated with parallel effects at the RNA level. Incorporation of (35)S-methionine into cellular proteins over a 4-h period starting 20 h after transfer to defined culture media was not significantly reduced in 1.0xcompared with 5.0x amino acid media, although rates under both of these conditions were significantly higher than those seen in 0.2xamino acid media (P<0.01). The lack of correspondence between the dose-dependent effects of amino acid supply on cellular protein synthesis and those on basal and GH-stimulated IGF-I production, suggests that amino acid supply modulates IGF-I production through selective mechanisms. Steady state levels of the CCAAT/enhancer-binding protein beta (C/EBPbeta) isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP) were determined by Western blotting. When levels of LAP were expressed relative to LIP levels in the same extracts, a significant decrease in the LAP:LIP ratio was observed in response to amino acid limitation (P<0.05). These data strengthen earlier arguments that synergistic interaction between the effects of amino acids and GH on hepatic IGF-I gene expression underlie nutrition-dependent changes in circulating IGF-I titres. The association between these effects and altered levels of C/EBPbeta isoforms suggests that CCAAT/enhancer mediated control of IGF-I gene expression may be involved in this phenomenon.

Effect of ambient temperature during 1st day of life on thermoregulation in lambs delivered by cesarean section.

We examined the effect of delivering near-term twin lambs by cesarean section into a warm (30 degrees C) or cool (15 degrees C) ambient temperature on the control of thermoregulation. Heat production was measured 20-30 h after birth during non-rapid-eye-movement sleep at 29 and 14 degrees C. At 29 degrees C there was no difference in heat production between groups, but at 14 degrees C cool-delivered (CD) lambs exhibited a 62% greater metabolic response. Irrespective of delivery temperature, 15 of the 18 lambs used shivering thermogenesis during cold exposure, indicating a reduction in the ability to use nonshivering thermogenesis in brown adipose tissue (BAT). Mean plasma concentrations of thyroxine and triiodothyronine were 35 and 45% greater, respectively, in CD lambs than in warm-delivered lambs. The level of guanosine 5'-diphosphate binding in BAT was lower than in normally delivered lambs and was not different between CD and warm-delivered lambs. Cesarean section delivery prevents the rise in BAT thermogenic activity, which results in an increased reliance on shivering thermogenesis to maintain colonic temperature. Under these conditions, delivery into a cool environment increases the plasma concentration of thyroid hormones, which benefits the neonate by enabling a greater thermogenic response via shivering.

Chronic cold exposure has no effect on brown adipose tissue in newborn lambs born to well-fed ewes.

It has been previously shown in twin-bearing ewes fed only 60% of their metabolizable energy requirements for late pregnancy that chronic cold exposure induced by winter shearing of the ewes results in larger lambs with more brown adipose tissue. This effect appears to be primarily due to prevention of a decline in fetal body and tissue weights between 145 days' gestation and 2 h after birth (i.e. 147 days' gestation) in lambs born to underfed shorn ewes. The present study therefore examined the impact, in ewes that were well fed (i.e. received 100% of their metabolizable energy requirements) during the final month of gestation, of chronic cold exposure induced by winter shearing on lamb birthweight and perirenal adipose tissue composition as measured 2 h after birth. Perirenal adipose tissue was analysed for its thermogenic activity (i.e. GDP binding to mitochondria) and catecholamine content. These observations were combined with similar measurements made in near-term (i.e. 145 days' gestation) fetuses sampled from well-fed unshorn ewes. There was no difference between lambs born to shorn or unshorn ewes with respect to birthweight or perirenal adipose tissue weight and composition. Perirenal adipose tissue weight was higher in lambs born to unshorn ewes than in fetuses. The thermogenic activity of adipose tissue was 2-fold higher in lambs born to unshorn ewes compared with 145-day-old fetuses. Epinephrine was detectable only at very low levels in fetal perirenal adipose tissue, increasing 10-fold after birth, with no difference between lambs born to shorn or unshorn ewes. In newborn lambs, plasma growth hormone concentration was lower and insulin concentration higher in shorn compared with unshorn groups. In conclusion, chronic cold exposure induced by winter shearing had no effect on brown adipose tissue development in lambs born to well-fed ewes.

Effect of maternal glucose infusion on brown adipose tissue and liver development in the neonatal lamb.

The effect of maternal glucose infusion over the final 5-7 days of gestation in under-fed ewes on perirenal brown adipose tissue (BAT) and liver development in lambs over the first month of neonatal life was examined. During glucose infusion, higher maternal plasma concentrations of glucose and thyroid hormones, and lower plasma concentrations of non-esterified fatty acids and 3-hydroxybutyrate were observed, compared with saline-infused controls. These differences were not observed 1-1.5 h before parturition when plasma concentrations of glucose, lactate, cortisol and thyroid hormones all increased in control ewes. Lamb birthweight and liver and BAT weights were similar between groups, but lambs born to glucose-infused ewes had a higher hepatic glycogen content and greater iodothyronine 5'deiodinase activities in liver and BAT. The norepinephrine, epinephrine and dopamine contents were also greater in BAT sampled from lambs born to glucose infused ewes. Three lambs born to glucose-infused ewes failed to survive beyond the second week of life and exhibited abnormally low plasma triiodothyronine concentrations. It is concluded that maternal glucose infusion stimulates development of the fetal sympathetic nervous system during late gestation but this adaptation does not appear to improve postnatal survival.

Brown adipose tissue and liver development during early postnatal life in hand-reared and ewe-reared lambs.

This study examined the effects of modest changes in ambient temperature in hand-reared lambs (experiment one) and in ewe-reared lambs (experiment two). Lambs were killed at either 8 or 31 days of age and perirenal adipose tissue was identified as being brown adipose tissue (BAT) from measurements of thermogenic activity (i.e. GDP binding to uncoupling protein in isolated mitochondria) or thermogenic capacity (i.e. detection of uncoupling protein by immunoblotting). In addition, type I and II iodothyronine 5' monodeiodonase (5'MDI) activities were assayed in perirenal adipose tissue, plus type I 5'MDI activity in liver. Plasma samples were also taken for measurements of glucose, lactate, insulin, triiodothyronine (T3) and thyroxine (T4) concentrations. In experiment one, lambs were hand-reared at either warm (WR; 25 degrees C) or cool (CR; 10-15 degrees C) ambient temperatures. Mean growth rate over the first 8 days of life in CR lambs was 88 g/day and increased to 128 g/day over the first month of life. Growth rate in WR lambs was constant at 141 g/day. Thermogenic activity of BAT was significantly higher in CR than WR lambs, but total weight and tissue lipid content of perirenal adipose tissue were significantly lower in the CR group. In both WR and CR lambs, the thermogenic activity of BAT fell by an average of 71% between 8 and 31 days. At 31 days of age, uncoupling protein in mitochondria could be detected only by immunoblotting in adipose tissue sampled from CR lambs. There was no effect of ambient temperature on type I or type II 5'MDI activity in BAT or liver; it decreased in adipose but not liver tissue between 8 and 31 days of age. The plasma concentrations of glucose, insulin and T3 tended to decline with age in CR but not in WR lambs. In ewe-reared lambs perirenal adipose tissue weight and tissue lipid content more than doubled between 8 and 31 days of age, but the level of GDP binding decreased from 85 to 5 pmol/mg mitochondrial protein over this period. Liver weight increased by 55% between 8 and 31 days of age, but hepatic 5'MDI activity remained unchanged. The plasma concentrations of T3, T4 and lactate, but not glucose or insulin, increased between 8 and 31 days of age. It is concluded that hand-rearing lambs at a cool ambient temperature significantly delays postnatal development, to the extent that BAT characteristics are retained. Ewe-rearing lambs enhances the rate at which BAT adopts the characteristics of white adipose tissue, and it prevents the postnatal decline in plasma concentrations of thyroid hormones.

Effect of fetal thyroidectomy on brown adipose tissue and thermoregulation in newborn lambs.

The effect of fetal thyroidectomy on thermoregulation in newborn lambs was investigated. Seven of 14 lambs born normally at term were thyroidectomized at Day 127 of gestation. Colonic temperature and oxygen consumption were measured during non-rapid eye movement sleep 6-45 h after birth. All lambs were then killed and perirenal brown adipose tissue was sampled for measurement of thermogenic activity (guanosine diphosphate binding), uncoupling protein and lipid contents. Thyroidectomized lambs tended to have a mean colonic temperature 2.35 degrees C lower (P = 0.067) than controls and two became hypothermic (i.e. colonic temperature < 35 degrees C). Thyroidectomized lambs exhibited lower rates of oxygen consumption (P = 0.05) and an increased incidence of shivering thermogenesis. The perirenal adipose tissue of these lambs had a lower thermogenic activity (P < 0.01), less uncoupling protein (P < 0.01) and higher lipid content (P = 0.072) compared with intact controls. It is concluded that fetal thyroidectomy results in a decreased ability of newborn lambs to utilize nonshivering thermogenesis in brown adipose tissue as well as increasing the incidence of hypothermia. These changes are associated with decreased synthesis of uncoupling protein and functional development of brown adipose tissue in the late gestation fetus.

Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro.

To determine whether serum supplementation influenced fatty acid content of bovine blastocysts and whether vitamin E addition to culture medium containing serum could improve development in vitro, cleaved eggs were cultured in synthetic oviduct fluid supplemented with bovine serum albumin (BSA, 0.4% w/v, fraction V) (SVBSA), fetal calf serum (FCS, 10% v/v) (SFCS) or FCS (10% v/v) plus 100 micro M vitamin E (SFCS + E). Blastocyst yields were recorded and fatty acid composition was determined by gas chromatography. Day 7 blastocysts were incubated with [2-(14)C] pyruvate for 3 h and then fixed for cell counts. Yields of good quality blastocysts were greatest from cleaved eggs cultured in serum-free conditions (P < 0.01). In the presence of serum, supplementation with vitamin E increased both total and good quality blastocyst yields (P < 0.01). Presence of serum increased fatty acid content (mean +/- SEM) of blastocysts (SVBSA v. SFCS = 57 +/- 2 v. 74 +/- 2 ng embryo(-1); P < 0.001). In contrast, pyruvate metabolism was greater in blastocysts produced without serum (27 +/- 3 v. 21 +/- 3 picomoles embryo(-1) 3h(-1); P < 0.01) but, on a per cell basis, no differences were detected. Addition of vitamin E to the serum-supplemented formulation did not alter either the fatty acid content (73 +/- 2 ng embryo(-1)) or pyruvate metabolism index (19 +/- 1 pmol embryo(-1) 3h(-1)) of SFCS + E blastocysts. Thus, despite lipid accumulation, supplementary vitamin E improved blastocyst yields in embryos exposed to serum.

Effect of short term intravenous infusion of noradrenaline on energy metabolism in shorn and unshorn pregnant sheep.

The effect of a three hour intravenous noradrenaline (NA) infusion, at a dose of 0.8 micrograms min-1 kg-1 bodyweight, on energy metabolism was studied in five pairs of shorn and unshorn pregnant ewes. The concentration of oxygen and carbon dioxide in blood was significantly higher in shorn animals during saline infusion, but this difference between shorn and unshorn groups was removed by NA infusion. Significant increases in the plasma concentrations of glucose, lactate, non-esterified fatty acids (NEFA), 3-hydroxybutyrate and glycerol occurred in both shorn and unshorn animals during NA infusion. There was a 48 per cent larger rise in the plasma NEFA concentration during NA infusion in the unshorn group, suggesting that the lipolytic effect of exogenous NA is greater in these animals. NA infusion had no effect on the plasma concentration of thyroid hormones in both groups of ewes, while growth hormone levels fell and cortisol increased in all animals. The plasma insulin concentration was significantly reduced during NA treatment in the unshorn group, but was unchanged in shorn animals. These results may be a consequence of sympathetic activity already being elevated in shorn ewes due to chronic exposure to an environmental temperature below the thermoneutral zone.

Effects of isoproterenol infusion on the hindlimb metabolism of growing wether lambs.

The effect of a mixed beta 1/beta 2-adrenergic agonist, isoproterenol, on hindlimb metabolism was studied in growing wether lambs using arteriovenous difference and blood flow rate techniques. Isoproterenol (48 micrograms kg-1 d-1), or saline, was infused into a jugular vein of five wether lambs (30 to 35 kg) for five days and samples taken on the fifth day of treatment. Infusion of isoproterenol significantly increased blood flow, oxygen uptake and tyrosine uptake across the hindlimb. Hindlimb non-esterified fatty acid uptake was increased but not significantly (P = 0.11) and arterial growth hormone concentration was not altered by isoproterenol infusion. Results suggest that beta-adrenergic agonists promote lean tissue deposition by increased muscle blood flow rate and amino acid uptake.

Synergism between cAMP and PPARγ Signalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes.

Expression of the brown adipocyte-specific gene, uncoupling protein 1 (UCP1), is increased by both PPARγ stimulation and cAMP activation through their ability to stimulate the expression of the PPAR coactivator PGC1α. In HIB1B brown preadipocytes, combination of the PPARγ agonist, rosiglitazone, and the cAMP stimulator forskolin synergistically increased UCP1 mRNA expression, but PGC1α expression was only increased additively by the two drugs. The PPARγ antagonist, GW9662, and the PKA inhibitor, H89, both inhibited UCP1 expression stimulated by rosiglitazone and forskolin but PGC1α expression was not altered to the same extent. Reporter studies demonstrated that combined rosiglitazone and forskolin synergistically activated transcription from a full length 3.1 kbp UCP1 luciferase promoter construct, but the response was only additive and much reduced when a minimal 260 bp proximal UCP1 promoter was examined. Rosiglitazone and forskolin in combination were able to synergistically stimulate promoters comprising of tandem repeats of either PPREs or CREs. We conclude that rosiglitazone and forskolin act together to synergistically activate the UCP1 promoter directly rather than by increasing PGC1α expression and by a mechanism involving cross-talk between the signalling systems regulating the CRE and PPRE on the promoters.