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JE Eckert, KL Gatford, BG Luxford, RG Campbell and PC Owens

Birth weight is a determinant of blood leptin concentrations in adults. Since nutrition during pregnancy can affect birth weight, the hypothesis that feed intake during pregnancy alters leptin expression in progeny was examined. Leptin mRNA was measured in subcutaneous adipose tissue and leptin protein was measuredin blood plasma from 59 day old female pigs whose mothers were fed at the same restricted rate except that half were permitted to consume 35% more feed during the second quarter of pregnancy. Leptin mRNA abundance in adipose tissue (P=0.015) and plasma leptin concentration (P=0.01) were higher in progeny from mothers provided with more feed. Body weight at birth was negatively correlated with the abundance of leptin mRNA in subcutaneous fat at 59 days of age (P=0.01). This study shows for the first time that maternal nutrition during pregnancy programs postnatal leptin expression in offspring.

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KL Gatford, AR Egan, IJ Clarke and PC Owens

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SJ Bernard, I Yuen, C McMillen, ME Symonds and PC Owens

Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to b-actin mRNA in fetal perirenal adipose tissue increased (P<0.02) with gestation, being higher at 144 d (0.73 +/- 0. 10, n=5) than at 90-91 d (0.40 +/- 0.08, n=6) or 125 d (0.40 +/- 0. 04, n=5) gestation (term approximately 147- 150 d). There was a positive relationship between relative abundance of leptin mRNA (y) and fetal body weight (x)between 90 and 144 d gestation (r 2 =0.27, P<0.01). The slope of the linear dependence of leptin mRNA on fetal weight was 15-fold greater (P<0.001) at 90-91d (y = 2.81x - 1.1, n=6, r 2 =0.71, P<0.025) than between 125-144 d gestation (y = 0.195x - 0.15, n=16, r 2 =0.39, P<0.01). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate.

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SE Bastian, AJ Dunbar, IK Priebe, PC Owens and C Goddard

Betacellulin, a member of the epidermal growth factor (EGF) family, was originally isolated and identified from the conditioned medium from a murine pancreatic beta-cell carcinoma cell line. Recently, we isolated bovine betacellulin from a growth factor enriched cheese whey extract, but there is no information on the presence of betacellulin in other biological fluids. We have cloned the cDNA for bovine betacellulin, produced recombinant betacellulin and shown that it has a similar potency to the purified native molecule in stimulating the proliferation of Balb/c3T3 fibroblasts. We have produced a polyclonal antiserum to bovine betacellulin which did not cross-react with EGF or transforming growth factor-alpha (TGF-alpha). The antibody was used in a homologous RIA that was able to detect betacellulin in pooled bovine colostrum sampled during the first 3 days after calving (2.30+/-0.11 ng/ml mean+/-s.e.m.; n=6), in bovine milk soluble fraction (1.93+/-0.64 ng/ml mean+/-s.e.m.; n=5) and in bovine cheese whey (2.59+/-0.16 ng/ml mean+/-s.e.m.; n=3). The betacellulin concentration in foetal bovine serum (FBS) (3.68+/-0.59 ng/ml mean+/-s.e.m.; n=6) greatly exceeded that of betacellulin in serum from male calves 1 and 5 weeks of age (0.53+/-0.15 ng/ml and 0.70+/- 0.09 ng/ml respectively; mean+/-s.e.m.; n=9). Betacellulin measured in the serum of these same animals when aged between 27 and 43 weeks was below the detection limits of the RIA. Sera from 10 out of 36 unmated heifers contained betacellulin levels within the detection limits of the assay (0.433+/-0.06 ng/ml mean+/-s.e.m.; n=10). The presence of betacellulin in bovine colostrum and milk suggests that it plays a role in the growth and development of the neonate and/or mammary gland function. The results also show that betacellulin is undetectable in the castrated adult male circulation. Additionally, although present in very low amounts, serum betacellulin could be under hormonal regulation in the female, since betacellulin was detected in sera from 27% of the unmated heifers examined in this study. The high levels of betacellulin detected in FBS relative to newborn and adult serum suggests a possible endocrine role for this growth factor in the bovine foetus.

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BS Yuen, IC McMillen, ME Symonds and PC Owens

Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to beta-actin mRNA in fetal perirenal adipose tissue increased (P<0.02) with gestation, being higher at 144 d (0.73+/-0. 10, n=5) than at 90-91 d (0.40 +/- 0.08, n=6) or 125 d (0.40 +/- 0. 04, n=5) gestation (term approximately 147- 150 d). There was a positive relationship between relative abundance of leptin mRNA (y) and fetal body weight (x) between 90 and 144 d gestation (r(2) =0.27, P<0.01). The slope of the linear dependence of leptin mRNA on fetal weight was 15-fold greater (P<0.001) at 90-91d (y = 2.81x - 1.1, n=6, r(2) =0.71, P<0.025) than between 125-144 d gestation (y = 0.195x - 0.15, n=16, r(2) =0.39, P<0.01). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate.

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A Sohlstrom, A Katsman, KL Kind, PA Grant, PC Owens, JS Robinson and JA Owens

The effect of fasting (17-18 h) versus food restriction (70% for 80 +/- 13 days) on the IGF-IGF binding protein (BP) axis in female guinea pigs was studied and related to body weight, weight gain and food conversion efficiency. Circulating IGF-I was reduced in the fasted (13%) and food-restricted (50%) animals. IGF-II was only decreased (61%) in the food-restricted group. There was no effect of fasting on IGFBP-1 to -4 while IGFBP-1, -3 and -4 were reduced by 56%, 60% and 44% respectively, and IGFBP-2 increased by 72%, in the food-restricted group. Food restriction reduced the relative sizes of fat depots, spleen, liver, thymus and heart, increased those of adrenals, kidneys, pancreas, gastrointestinal tract, M. Biceps, M. Soleus and brain while those of uterus, lungs, thyroids and M. Gastrocnemius were unchanged. IGFBP-1 and -2 were negatively correlated to weight gain and food conversion efficiency in the ad libitum-fed group, while IGF-I, -II, IGFBP-1, -3 and -4 were positively correlated to body weight, weight gain and food conversion efficiency in the food-restricted group. The results show that acute and chronic food restriction have different consequences for the IGF-IGFBP axis. Furthermore, IGF-II as well as IGF-I are implicated in the control of body weight, weight gain and food conversion efficiency under conditions of restricted nutrition. Finally, IGFBP-1 and -2 may have different roles during chronic undernutrition compared with unrestrained nutrition in adult life.

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KL Gatford, JA Owens, RG Campbell, JM Boyce, PA Grant, MJ De Blasio and PC Owens

Circulating growth hormone (GH) concentrations increase in pregnancy and administration of GH during early-mid pregnancy increases fetal growth in well-fed pigs. To determine whether increased maternal GH could promote fetal growth when feed availability is restricted, fifteen cross-bred primiparous sows (gilts) were fed at approximately 30% of ad libitum intake, from mating onwards and were injected daily i.m. with recombinant porcine GH (pGH) at doses of 0, 13.4+/-0.3 and 25.6+/-0.5 microg/kg live weight from day 25 to day 51 of pregnancy (term approximately 115 days). Treatment with pGH increased maternal backfat loss between day 25 and day 51 of pregnancy, and increased maternal plasma IGF-I concentrations measured at day 51 of pregnancy. Fetal body weight, length and skull width at day 51 of pregnancy were increased by maternal treatment with pGH. Fetal plasma glucose concentrations were increased and maternal/fetal plasma glucose concentration gradients were decreased by maternal pGH treatment at 13.4, but not 25.6 microg/kg.day. Fetal plasma concentrations of urea were decreased by both levels of pGH treatment. Overall, fetal weight was negatively correlated with fetal plasma concentrations of urea, positively correlated with maternal plasma alpha-amino nitrogen concentrations and unrelated to glucose concentrations in either maternal or fetal plasma. This suggests that the availability of amino acids, not glucose, limits fetal growth in the first half of pregnancy in underfed gilts, and that maternal GH treatment may improve amino acid delivery to the fetus.

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KL Gatford, KJ Quinn, PE Walton, PA Grant, BJ Hosking, AR Egan and PC Owens

The ontogeny of the IGF endocrine system was investigated in 15 young lambs before and after weaning at 62 days of age. Before weaning, plasma IGF-I concentrations were higher in rams than ewes, and plasma concentrations of IGF-II and IGF-binding protein-3 (IGFBP-3) also tended to be higher in rams than in ewes. Feed intake of ewes and rams was restricted after weaning to remove sex differences in feed intake. Plasma concentrations of IGF-I and IGFBP-3 did not differ between rams and ewes at 100 days of age, but plasma IGF-II was higher in rams than in ewes at this time. Since circulating concentrations of GH were higher in rams than in ewes at 100 days of age, this implies that the restricted feed intake blocked the IGF-I and IGFBP-3 responses to GH. We conclude that sex differences in circulating IGF-I and IGFBP-3 concentrations in the growing lamb alter with age, and are not present when nutrition is restricted.

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O Gajanandana, K Irvine, PA Grant, GL Francis, SE Knowles, J Wrin, JC Wallace and PC Owens

Long-Arg3-IGF-I (LR3IGF-I) is a synthetic analog of IGF-I that has much lower affinity for IGF-binding proteins than do native IGFs-I and -II. Comparisons of the effects of LR3IGF-I with those of IGFs-I and-II in in vitro and in vivo studies have proved useful in defining the functions of IGF-binding proteins. We have developed a sensitive noncompetitive nonisotopic assay of LR3IGF-I. Mouse IgG 1A7-F5-E5 binds an epitope that contains the substituted arginine3 in LR3IGF-I and was used as the solid phase antibody. The solution phase antibody was a rabbit immunoglobulin Nelson which binds to an epitope that is common to IGF-I and LR3IGF-I. The ELISA system was able to detect as little as 50 pg LR3IGF-I in 100 microliters and the native peptides IGFs-I and -II have less than 0.01% activity. Blood plasma from animals treated with pharmacologically active doses of this growth factor analog could be diluted 33.3-fold before assay, at which concentrations plasma had no significant effect on the assay. The ELISA response to LR3IGF-I was unaffected by the presence of IGF-binding proteins. The intra-assay and interassay coefficients of variation are 2.8 and 7.3% respectively. Recovery of LR3IGF-I added to blood plasma was approximately 90%. The ELISA was used to measure LR3IGF-I concentrations in plasma of cows treated with a pharmacologically active dose of this peptide and the results were compared with those obtained by a previously established LR3IGF-I RIA that requires size exclusion chromatography of plasma under acidic conditions to eliminate IGF-binding protein artefacts from the RIA. There was a positive correlation between results obtained by the two assays. The LR3IGF-I ELISA permits discrimination between the exogenous synthetic IGF-I analog and the endogenous native IGFs-I and -II in animals treated with this growth factor without the need for radioiodination of LR3IGF-I and elimination of the requirement for extraction of plasma before assay.