Insensitivity of well-conditioned mature sheep to central administration of a leptin receptor antagonist.

Ruminants remain productive during the energy insufficiency of late pregnancy or early lactation by evoking metabolic adaptations sparing available energy and nutrients (e.g. higher metabolic efficiency and induction of insulin resistance). A deficit in central leptin signaling triggers these adaptations in rodents but whether it does in ruminants remains unclear. To address this issue, five mature ewes were implanted with intracerebroventricular (ICV) cannula in the third ventricle. They were used in two experiments with an ovine leptin antagonist (OLA) when well-conditioned (average body condition score of 3.7 on a 5 point scale). The first experiment tested the ability of OLA to antagonize leptin under in vivo conditions. Ewes received continuous ICV infusion of artificial cerebrospinal fluid (aCSF), ovine leptin (4 µg/h) or the combination of ovine leptin (4 µg/h) and its mutant version OLA (40 µg/h) for 48 h. Dry matter intake (DMI) was measured every day and blood samples were collected on the last day of infusion. ICV infusion of leptin reduced DMI by 24% (P < 0.05), and this effect was completely abolished by OLA co-infusion. A second experiment tested whether a reduction in endogenous leptin signaling in the brain triggers metabolic adaptations. This involved continuous ICV infusions of aCSF or OLA alone (40 µg/h) for 4 consecutive days. The infusion of OLA did not alter voluntary DMI over the treatment period or on any individual day. OLA did not affect plasma variables indicative of insulin action (glucose, non-esterified fatty acids, insulin and the disposition of plasma glucose during an insulin tolerance test) or plasma cortisol, but tended to reduce plasma triiodothyronine and thyroxine (P < 0.07). Overall, these data show that a reduction of central leptin signaling has little impact on insulin action in well-conditioned mature sheep. They also raise the possibility that reduced central leptin signaling plays a role in controlling thyroid hormone production.

Effect of nutrition on the GH responsiveness of liver and adipose tissue in dairy cows.

Dairy cows enter a period of energy insufficiency after parturition. In liver, this energy deficit leads to reduced expression of the liver-specific GH receptor transcript (GHR1A) and decreased GHR abundance. As a consequence, hepatic processes stimulated by GH, such as IGF-I production, are reduced. In contrast, adipose tissue has been assumed to remain fully GH responsive in early lactation. To determine whether energy insufficiency causes contrasting changes in the GH responsiveness of liver and adipose tissue, six lactating dairy cows were treated for 4 days with saline or bovine GH when adequately fed (AF, 120% of total energy requirement) or underfed (UF, 30% of maintenance energy requirement). AF cows mounted robust GH responses in liver (plasma IGF-I and IGF-I mRNA) and adipose tissue (epinephrine-stimulated release of non-esterified fatty acids in plasma, IGF-I mRNA, and p85 regulatory subunit of phosphatidylinositol 3-kinase mRNA). Reductions of these responses were seen in the liver and adipose tissue of UF cows and were associated with decreased GHR abundance. Reduced GHR abundance occurred without corresponding reductions of GHR1A transcripts in liver or total GHR transcripts in adipose tissue. In contrast, undernutrition did not alter the abundance of proteins involved in the early post-receptor signaling steps. Thus, a feed restriction reproducing the energy deficit of early lactation depresses GH actions not only in liver but also in adipose tissue. It remains unknown whether a similar reduction of GH action occurs in the adipose tissue of early lactating dairy cows.

Effect of feed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, testosterone, and on sexual development in bulls.

The objective of the present study was to evaluate the effects offeed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, and testosterone, and on sexual development in bulls. Eight beef bull calves received a control diet from 10 to 70 weeks of age. An additional 16 calves had restricted feed (75% of control) from 10 to 26 weeks of age (calfhood), followed by either control or high nutrition (n=8/group) during the peripubertal period until 70 weeks of age. Restricted feed during calfhood inhibited the hypothalamic GnRH pulse generator, reduced the pituitary response to GnRH, impaired testicular steroidogenesis, delayed puberty, and reduced testicular weight at 70 weeks of age, regardless of the nutrition during the peripubertal period. Restricted feed reduced serum IGF-I concentrations, but concentrations of leptin, insulin, and GH were not affected. In conclusion, restricted feed during calfhood impaired sexual development in bulls due to adverse effects on every level of the hypothalamus-pituitary-gonad axis and these effects were not overcome by supplemental feeding during the peripubertal period. Furthermore, based on temporal associations, the effects of restricted feed on the hypothalamus-pituitary-gonad axis might be mediated by serum IGF-I concentrations. These results supported the hypotheses that the pattern of LH secretion during the early gonadotropin rise during calfhood is the main determinant of age of puberty in bulls and that gonadotropin-independent mechanisms involved in testicular growth during the peripubertal period are affected by previous LH exposure.

Localisation of glucose transport in the ruminant placenta: implications for sequential use of transporter isoforms.

The facilitative glucose transporters 1 and 3 are the major routes for glucose transport across placental membranes. Using light and electron microscope immunocytochemistry on acrylic sections this study shows a similar pattern of expression from mid to late pregnancy in all four ruminants examined [cow, deer, ewe and goat]. GT1 and GT3 are localised on different membrane layers of the synepitheliochorial placental barrier and glucose must utilise both isoforms sequentially to pass from the maternal to fetal circulations. It is suggested that this arrangement is designed to support the high glucose utilisation by the multilayered placenta in the ruminant.

Demonstration of a role for insulin in the regulation of leptin in lactating dairy cows.

In lactating dairy cows, the onset of negative net energy balance (EB) at parturition causes a reduction in plasma leptin and is also associated with increased concentration of growth hormone (GH) and decreased concentration of insulin. These observations raise the possibility that opposite changes in plasma insulin and GH are partly responsible for reduced plasma leptin. To test this hypothesis, we first examined the effects of undernutrition without the confounding influence of parturition by using late lactating dairy cows fed 120% of their nutrient requirements or restricted to 33% of maintenance requirements. Plasma leptin was reduced within 24 h of feed restriction, and was associated with increased plasma GH and decreased plasma insulin. Complete food deprivation for 48 h caused similar changes in the plasma concentration of leptin. To determine if an elevation in GH is responsible for the fall in plasma leptin, dairy cows were treated with excipient or bovine somatotropin in early lactation or in late lactation. Growth hormone treatment had no significant effect on plasma leptin irrespective of stage of lactation. Finally, the effects of insulin were studied by performing euglycemic hyperinsulinemic clamps in mid-lactating dairy cows. After 96 h of hyperinsulinemia, plasma leptin was increased significantly. These data indicate that insulin regulates plasma leptin in lactating dairy cows. They also suggest that, in undernourished lactating dairy cows, reduced plasma insulin could account for a portion of the decline in plasma leptin but that elevated plasma GH is unlikely to have a major effect.

Nutritional and developmental regulation of plasma leptin in dairy cattle.

Leptin is thought to play a critical role in regulating energy metabolism throughout mammalian life. In growing dairy cattle, plasma leptin has been proposed as a partial mediator of the effects of nutrition on reproductive and mammary development. However, the developmental stage at which the plane of nutrition increases plasma leptin has not been well defined. Further, it is unknown whether the onset of puberty is affected by plasma leptin concentration in dairy cattle. To investigate these questions, two studies were performed. In the first study, neonatal calves were fed a milk replacer at levels supporting an average daily gain of 570 g/d (L) or 1210 g/d (H). Weekly blood samples were obtained until slaughter at 105 kg of body weight. Plasma leptin and adiposity remained constant in the L calves, but started to increase by the third week of age in the H calves. In the second study, 3- to 5-mo-old heifers were fed a total mixed ration supplemented with either calcium salts of palm fat or conjugated linoleic acids at levels sustaining an average daily gain of approximately 1.0 kg/d. Blood samples were obtained until the third postpubertal luteal phase. The fat source had no effects on growth parameters, body composition, age at puberty, or plasma leptin. Therefore, plasma leptin was reanalyzed as a function of age from start of treatment until slaughter. The plasma concentration of leptin remained nearly constant at 2.3 ng/ml until 1 yr of age, when a rise in plasma leptin became obvious. Puberty occurred with equal frequency either around 1 yr of age when plasma leptin was nearly constant or later when leptin was rising rapidly. We conclude that plasma leptin is regulated by nutrition in early postnatal life, but that a sudden increase in plasma leptin is not required for the onset of puberty in dairy cattle.

Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.

Dairy cows suffer from an intense energy deficit at parturition due to the onset of copious milk synthesis and depressed appetite. Despite this deficit, maternal metabolism is almost completely devoted to the support of mammary metabolism. Evidence from rodents suggests that, during periods of nutritional insufficiency, a reduction in plasma leptin serves to co-ordinate energy metabolism. As an initial step to determine if leptin plays this role in periparturient dairy cows, changes in the plasma concentration of leptin were measured during the period from 35 days before to 56 days after parturition. The plasma concentration of leptin was reduced by approximately 50% after parturition and remained depressed during lactation despite a gradual improvement in energy balance; corresponding changes occurred in the abundance of leptin mRNA in white adipose tissue. To determine whether negative energy balance caused this reduction in circulating leptin, cows were either milked or not milked after parturition. Absence of milk removal eliminated the energy deficit of early lactation, and doubled the plasma concentration of leptin. The plasma concentration of leptin was positively correlated with plasma concentrations of insulin and glucose, and negatively correlated with plasma concentrations of growth hormone and non-esterified fatty acids. In conclusion, the energy deficit of periparturient cows causes a sustained reduction in plasma leptin. This reduction could benefit early lactating dairy cows by promoting a faster increase in feed intake and by diverting energy from non-vital functions such as reproduction.

Maternal leptin is elevated during pregnancy in sheep.

Maternal plasma leptin is elevated during pregnancy in several species, but it is unclear to what extent this elevation reflects changes in adiposity or energy balance. Therefore, Karakul ewes (n = 8) were fed to minimize changes in maternal energy status over the pregnancy-lactation cycle. They were studied 20-40 d before breeding and during mid pregnancy (d 50-60 post coitus [PC]), late pregnancy (d 125-135 PC) and early lactation (d 15-22 post partum). Consistent with the maintenance of near energy equilibrium in nongravid maternal tissues, maternal body weight was increased only during late pregnancy when the weight of the conceptus became significant and plasma concentrations of insulin, NEFA and glucose did not vary with physiological state. In contrast, maternal plasma leptin concentration rose from 5.3 to 9.5 ng/mL between prebreeding and mid pregnancy and then declined progressively through late pregnancy and early lactation. Leptin gene expression increased 2.3 fold in maternal white adipose tissue (WAT) from prebreeding to mid pregnancy and declined to prebreeding levels during early lactation. To determine whether tissue response to insulin was involved in this effect, insulin tolerance tests were performed. The maternal plasma glucose response declined from prebreeding to early lactation, but was not correlated with either plasma leptin concentration or WAT leptin mRNA abundance. In conclusion, pregnancy causes an increase in the synthesis of leptin in sheep. This stimulation does not require increases in adiposity or energy balance and is unrelated to the ability of insulin to promote glucose utilization.

Time-dependent physiological regulation of ovine placental GLUT-3 glucose transporter protein.

We immunolocalized the GLUT-3 glucose transporter isoform versus GLUT-1 in the late-gestation epitheliochorial ovine placenta, and we examined the effect of chronic maternal hyperglycemia and hypoglycemia on placental GLUT-3 concentrations. GLUT-3 was limited to the apical surface of the trophoectoderm, whereas GLUT-1 was on the basolateral and apical surfaces of this cell layer and in the epithelial cells lining the placental uterine glands. GLUT-3 concentrations declined at 17-20 days of chronic hyperglycemia (P < 0.05), associated with increased uterine and uteroplacental net glucose uptake rate, but a normal fetal glucose uptake rate was observed. Chronic hypoglycemia did not change GLUT-3 concentrations, although uterine, uteroplacental, and fetal net glucose uptake rates were decreased. Thus maternal hyperglycemia causes a time-dependent decline in the entire placental glucose transporter pool (GLUT-1 and GLUT-3). In contrast, maternal hypoglycemia decreases GLUT-1 but not GLUT-3, resulting in a relatively increased GLUT-3 contribution to the placental glucose transporter pool, which could maintain glucose delivery to the placenta relative to the fetus when maternal glucose is low.

Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep.

Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of (125)I-labeled bovine leptin was linearly related (R(2)= 0.99) to the log of added bovine or ovine leptin between 0.1 to 2.0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of! nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.

Placental transport of nutrients and its implications for fetal growth.

Placental growth during early and mid-pregnancy has a powerful, constraining influence on fetal growth during late pregnancy. Studies involving surgical and environmental reduction of placental size in sheep have shown an associated reduction in capacity to transport oxygen, glucose and amino acids. Oxygen transport is limited by placental blood flow but transport of glucose and amino acids is determined by the abundance and activity of specific transport proteins. Glucose transporters include the GLUT1 and GLUT3 isoforms previously identified in brain and other tissues; systems for active transport of amino acids have been inferred but not characterized. Placental metabolism of glucose and amino acids has major effects both on the quantity of carbon and nitrogen delivered to the fetus, and on the composition of substrates involved. For example, the uteroplacental tissues consume more than 60% of uterine glucose uptake during late pregnancy, and the placenta substantially modifies the pattern of amino acids delivered to fetal blood. The placenta also participates in the array of metabolic adaptations of maternal and conceptus tissues to altered maternal nutrient supply. Placental capacity for glucose transport in moderately undernourished ewes is upregulated, partly by increased expression of the GLUT3 transport protein. During more severe glucose deprivation, placental transfer and fetal uptake of glucose are constrained in proportion with maternal supply, leading to fetal growth retardation.

Developmental increases in glucose transporter concentration in the sheep placenta.

To explore the molecular basis for the gestational increase in glucose transport capacity of the sheep placenta in vivo, placentas from twin-pregnant ewes at days 75, 110, and 140 postcoitus (n = 6/group) were analyzed for glucose transporter (GT) concentration. Concentration (pmol/mg protein) of D-glucose-inhibitable binding sites, measured by [3H]cytochalasin B binding analysis, increased 3.4 times from mid- to late pregnancy. Concurrently, abundance of GLUT-1 and GLUT-3 protein, measured by immunoblotting with specific polyclonal antibodies, increased 2.3 and 2.9 times, respectively, while abundance of GLUT-1 and GLUT-3 mRNA, measured by Northern blotting, increased 1.8 and 3.9 times, respectively. GLUT-4 protein was undetectable at all stages of pregnancy. Quantitative immunoblotting indicated that GLUT-1 accounted for 86.8 +/- 1.6% at day 75 and 56.1 +/- 4.1% at day 140 of total cytochalasin B binding sites. Thus increases in GT concentration explain much of the gestational increase in glucose transfer capacity observed in vivo. The gestational decline in relative contribution of GLUT-1 to cytochalasin binding, together with the greater developmental increases in GLUT-3 mRNA and protein, further suggests that the relative importance of GLUT-3 increases with gestational age.

Protein requirements of sheep in late pregnancy: partitioning of nitrogen between gravid uterus and maternal tissues.

The objective of this study was to quantify effects of maternal protein nutrition on N accretion or loss in conceptus and maternal tissues of ewes during late pregnancy. Ewes, pregnant with twins, were fed low (LP, 79 g CP/kg DM), medium (MP, 116 g CP/kg DM), or high (HP, 157 g CP/kg DM) protein diets, each with an estimated ME concentration of 2.7 Mcal/kg DM, between d 111 and 140 of pregnancy; all ewes had been fed the same diet (2.7 Mcal ME, 120 g CP/kg DM) for the previous 30 d (d 80 to 110). Dry matter intakes were varied (LP = 1.0, MP = 1.2, and HP = 1.4 kg/d) according to predicted energy costs of protein deposition for each diet. Nitrogen accretion was estimated by comparative slaughter (d 140 minus d 110) and by collection of excreta between d 120 and 130. Fresh weights of maternal and gravid uterine tissues were measured at slaughter, before proximate analysis of these components. Whole-body N retention was directly and linearly related to N intake, but efficiency of deposition of apparently absorbed N decreased linearly with increasing N intake (LP, .79; MP, .70; HP, .62). Nitrogen accretion in the gravid uterus, maternal viscera, and mammary gland was significantly less in LP than in MP or HP ewes. Nitrogen balance in maternal carcass tissues was linearly related to N intake, ranging from a negative value in LP ewes to a positive value in HP ewes (LP, -63 g; MP -39 g; HP, 55 g). These data provide the basis for estimating N requirements for protein accretion in the conceptus and in maternal tissues during late pregnancy. They also highlight the capacity of maternal carcass tissues to mobilize or deposit amino acids in response to variations in dietary protein supply.

Growth and metabolism of the ovine placenta during mid-gestation.

The timing and metabolic basis for the rapid increase then cessation of placental growth in sheep and the accompanying changes in tissue cellularity were defined in the present study. Placental growth proceeded rapidly from day 40 of gestation to an apex at day 75-80 with no change is tissue dry matter content observed thereafter to day 100. These macroscopic growth patterns are similar to those observed previously, but present results define an earlier apex in placental mass. Absolute growth rate of the placenta reached a maximum near day 55, as derived from the Gompertz equation, concomitant with a period of maximum hyperplastic growth between days 50 and 60. A rapid increase in DNA synthesis and tissue mass per nuclei number from day 40-50 was followed by proliferative growth to day 70. Net cellular proliferation apparently ceased by day 80 as indicated by the apex in DNA content and the beginning of a static, low rate of DNA synthesis that continued to day 100. Patterns of change in the fractional rates of protein synthesis (Ks) and accretion (Kg) were similar suggesting that changes in Ks explain much of the change in Kg. This study, through its identification of key phases in the cellular growth of the ovine placenta, has laid the foundation for future research on mechanisms of placental growth regulation.

Growth and accretion of energy and protein in the gravid uterus during late pregnancy in Holstein cows.

Multiparous Holstein cows (n = 18) were bred artificially to the same bull and then slaughtered at times ranging from 190 to 270 d postconception to assess accretion of energy, protein, fat, and ash by the conceptus. Wet weights, dry weights, and concentrations of energy, CP, crude fat, and ash were obtained for the following: fetus, combined amniotic and allantoic fluids, fetal membranes, cotyledons, caruncles, and uterine tissues. Rates of accumulation of these components in the gravid uterus (sum of all uterine contents) and fetus were described by linear or quadratic equations. Estimated rates of accretion of energy in the gravid uterus (i.e., conceptus) increased from 567 kcal/d at 190 d of gestation to 821 kcal/d at 270 d of gestation; corresponding rates of accretion of CP were 62 and 117 g/d. These daily rates represent net energy and protein requirements for conceptus growth during late pregnancy in mature Holstein cows. Conversion of predicted net energy to metabolizable energy requirements for conceptus growth, using the accepted efficiency factor of .14, yielded estimates that were consistent with current NRC recommendations. Factorial estimation of absorbed protein requirements is hampered by lack of precise information on the efficiency with which absorbed AA are deposited in conceptus tissues.

Pregnancy but not moderate undernutrition attenuates insulin suppression of fat mobilization in sheep.

Nonpregnant and late-pregnant ditocous ewes were fed either to maintain zero energy balance in maternal tissues (fed) or at 50% of this level (underfed) for several weeks. Plasma concentrations of nonesterified fatty acids (NEFA) and glycerol were measured under basal conditions and during infusion of various doses of insulin while maintaining euglycemia (hyperinsulinemic, euglycemic clamp technique). Pregnancy and undernutrition separately increased basal plasma NEFA concentration in an additive manner; plasma glycerol was increased by pregnancy but unaffected by undernutrition. The molar ratio of NEFA to glycerol was significantly greater in underfed ewes. Analysis of dose-response relations between plasma insulin and metabolites during insulin infusions showed that maximally insulin-suppressed concentrations of NEFA and glycerol were significantly greater in pregnant than in nonpregnant ewes but were unaffected by undernutrition. Neither pregnancy nor undernutrition affected the maximally insulin-suppressed NEFA to glycerol ratio, or the plasma insulin concentration for 50% maximal responses to insulin of plasma NEFA, plasma glycerol, or the plasma NEFA to glycerol ratio. Thus, even in ewes at or close to zero energy balance, pregnancy seems to reduce adipose responsiveness but not sensitivity to the antilipolytic effect of insulin. This is another manifestation of the normal development of insulin resistance in maternal tissues during late pregnancy.

Pregnancy and undernutrition alter glucose metabolic responses to insulin in sheep.

Nonpregnant and late-pregnant ditocous ewes were fed either to maintain zero energy balance in maternal tissues (fed) or at 50% of this level (underfed) for several weeks. Whole-body kinetics of glucose metabolism were measured under basal conditions, and the hyperinsulinemic, euglycemic clamp technique was used to define insulin-dose response profiles for several indices of whole-body glucose utilization, and for endogenous glucose production. Pregnancy increased and undernutrition decreased basal glucose entry rate (GER), glucose metabolic clearance rate (GMCR) and insulin-independent glucose utilization (IIGU). The consistent increment in IIGU of pregnant over nonpregnant ewes was comparable to previous estimates of uterine glucose uptake. Pregnancy resulted in higher plasma concentration for 50% maximal responses (ED50) to insulin of GER, GMCR, steady-state glucose infusion rate (SSGIR) to maintain euglycemia and insulin-dependent glucose utilization (IDGU). These changes were especially large in underfed pregnant ewes. Effects on the maximal response to insulin of these variables (Rmax) were relatively small (GMCR, IDGU) or nonsignificant (GER, SSGIR). Maximum insulin-induced suppression of endogenous glucose production was significantly lower due to undernutrition; neither Rmax nor ED50 for this response was affected by pregnancy. Insulin resistance in late-pregnant ewes is primarily due to decreased insulin sensitivity in (presumably) peripheral tissues, implying an alteration of receptor function or of early postreceptor signal transduction.

Relations between maternal and fetal plasma concentrations of placental lactogen and placental and fetal weights in well-fed ewes.

The concentration of ovine placental lactogen (oPL) in maternal plasma varies with litter size and nutritional status, making it difficult to compare these concentrations across studies. In this study, 27 Dorset and Finn-Dorset crossbred ewes with litters of known size and gestational age were used to relate concentrations of oPL in maternal plasma to placental and fetal weights. Fetal oPL concentrations also were correlated to these variables in 12 chronically catheterized singleton fetuses. The concentration of oPL in maternal plasma increased with increasing placental weight across litter sizes ranging from 1 to 3 (r = .716). When expressed per gram of placenta, oPL was greater (P less than .05) in those ewes carrying multiple fetuses. There was no correlation between maternal and fetal oPL in time-matched samples or in average values between individuals for ewes carrying singleton pregnancies. Within the singleton group, placental weight and fetal weight were well correlated (r = .761), as were the concentration of fetal plasma oPL and fetal weight (r = .699). Placental weight plus fetal oPL could explain 81% of the variation seen in fetal weight. These results imply that maternal and fetal oPL release are controlled independently and that fetal oPL affects fetal growth by a mechanism not directly related to placental size.