D. H. Keisler

Leptin Gene Expression, Circulating Leptin, and Luteinizing Hormone Pulsatility Are Acutely Responsive to Short-Term Fasting in Prepubertal Heifers: Relationships to Circulating Insulin and Insulin-Like Growth Factor I1

Abstract In the present study, we tested the hypothesis that short-term fasting would reduce leptin gene expression, circulating leptin, and LH pulsatility in prepubertal heifers in association with a decrease in circulating concentrations of insulin and insulin-like growth factor I (IGF-I). Twelve prepubertal crossbred heifers (mean ± SD = 315 ± 5 kg body weight) were assigned randomly to one of two treatments in two replicates: 1) control; normal feed consumption (n = 6) and 2) fasted; 48 h of total feed restriction (n = 6). Blood samples were collected at 15-min intervals for 8 h on Days 0 and 2 of the experiment and twice on Day 1. Subcutaneous fat samples were collected before treatment onset (Day −1) and at the end of the intensive blood sampling on Day 2. Acute feed restriction markedly reduced leptin mRNA in adipose tissue (P < 0.01) and circulating concentrations of leptin (P < 0.05), IGF-I (P < 0.01), and insulin (P = 0.05) as compared with controls on Day 2. Moreover, the treatment × day interaction (P < 0.076) and within-day contrasts (expressed as a percentage of Day 0 values) revealed that the mean frequency of LH pulses in the fasted group was lower (P < 0.06) than in controls on Day 2. Neither mean concentrations of growth hormone (GH) nor GH secretory dynamics were affected by acute feed restriction. Fasting-mediated decreases in leptin gene expression and circulating leptin, in association with reductions in secretion of IGF-I, insulin, and LH, provide a basis for investigating leptin as a hormone signaling energy status to the central reproductive axis in cattle.

Leptin as a predictor of carcass composition in beef cattle.

Our objective was to determine if serum concentrations of leptin could be used to predict carcass composition and merit in feedlot finished cattle. Two different groups of crossbred Bos taurus steers and heifers were managed under feedlot conditions near Miles City, MT. The first group consisted of 88 1/2 Red Angus, 1/4 Charolais, and 1/4 Tarentaise composite gene combination steers (CGC) harvested at the ConAgra processing facility in Greeley, CO. The second group (Lean Beef Project; LB) consisted of 91 F2 steers and heifers born to Limousin, Hereford, or Piedmontese by CGC F1 cows crossed to F1 bulls of similar breed composition and harvested at a local processing facility in Miles City, MT. Blood samples were collected approximately 24 h before harvest (CGC) or approximately 3 d before and at harvest (LB). No differences in serum concentrations of leptin were detected (P > 0.10) between Hereford, Limousin, or Piedmontese F2 calves nor between LB steers and heifers. Positive correlations (P < 0.01) existed between serum leptin and marbling score (r = 0.35 and 0.50), fat depth measured between the 12th and 13th rib (r = 0.34 and 0.46), kidney, pelvic, and heart fat (KPH) (r = 0.42 and 0.46), and quality grade (r = 0.36 and 0.49) in CGC and LB cattle, respectively. Serum leptin was also positively correlated with calculated yield grade for CGC steers (r = 0. 19; P = 0. 10) and LB cattle (r = 0.52; P < 0.01). Longissimus area was not correlated with serum leptin in CGC steers (r = 0.12; P > 0.10). However, a negative correlation existed between longissimus area and serum leptin in the LB cattle (r = -0.45; P < 0.01). Serum concentrations of leptin were significantly associated with carcass composition (marbling, back fat depth, and KPH fat) and quality grade in both groups of cattle studied and may provide an additional indicator of fat content in feedlot cattle.

Large-scale preparation of biologically active recombinant ovine obese protein (leptin)

Prokaryotic expression vector pMON3401 encoding full size A(‐1) ovine leptin was prepared by polymerase chain reaction (PCR) of previously described cDNA. E. coli cells transformed with this vector overexpressed large amounts of ovine leptin upon induction with nalidixic acid. The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on Q‐Sepharose and SP‐Sepharose columns, yielding two electrophoretically pure fractions (leptin‐Q and leptin‐SP), composed respectively of 90 and 95% of monomeric protein of the expected molecular mass of 16 kDa. The purified protein was capable of interacting with antibodies raised against GST‐ovine leptin and to bind specifically to ventromedial hypothalamus of ewes. The biological activity of both fractions resulting from proper renaturation was further evidenced by their ability to stimulate DNA synthesis in leptin‐sensitive BAF/3 cells transfected with a long form of human leptin receptor construct.

DIFFERENTIAL EFFECTS OF ESTROGEN AND PROLACTIN ON AUTOIMMUNE DISEASE IN THE NZB/NZW F1 MOUSE MODEL OF SYSTEMIC LUPUS ERYTHEMATOSUS

Estrogen and prolactin have been shown to modulate autoimmunity in the NZB/NZW F1 (B/W) mouse model of systemic lupus erythematosus (SLE). However, estrogen stimulates prolactin secretion. The goal of this study was to examine differential effects of estrogen and prolactin in the female B/W mouse model of SLE. B/W females were manipulated to create combinations of low and high concentrations of serum estrogen and prolactin. Hyperprolactinemic mice with either low or high serum estrogen levels had accelerated development of albuminuria at 24 and 32 weeks of age compared to normal and hypoprolatinemic mice. High estrogen/high prolactin mice also had a higher percentage of anti-DNA antibodies compared to mice in the low estrogen/low prolactin and the high estrogen/low prolactin groups. IgG levels were not significantly different between groups. Mean survival was shortest in the high estrogen/high prolactin group (34 1.0 weeks) and longest in the high estrogen/low prolactin group (42 1.2 weeks; P < 0.05). High levels of serum estrogen were associated with depressed in vitro lymphoproliferation and IL-2 production. This study suggests that high prolactin levels in either high or low serum estrogen states are associated with accelerated autoimmunity in the B/W mouse. This study further demonstrates that high estrogen levels do not accelerate murine SLE when the prolactin-stimulating property of estrogen is suppressed by bromocriptine. Further investigation of hormonal interactions in autoimmunity will provide a better understanding of hormonal immunoregulation and, perhaps, lead to improved clinical application of hormonal immunomodulation.

Influence of maternal pre-pregnancy body composition and diet during early-mid pregnancy on cardiovascular function and nephron number in juvenile sheep.

The prenatal diet can program an individuals cardiovascular system towards later higher resting blood pressure and kidney dysfunction, but the extent to which these programmed responses are directly determined by the timing of maternal nutritional manipulation is unknown. In the present study we examined whether maternal nutrient restriction targeted over the period of maximal placental growth, i.e. days 28-80 of gestation, resulted in altered blood pressure or kidney development in the juvenile offspring. This was undertaken in 6-month-old sheep born to mothers fed control (100-150 % of the recommended metabolisable energy (ME) intake for that stage of gestation) or nutrient-restricted (NR; 50 % ME; n 6) diets between days 28 and 80 of gestation. Controls were additionally grouped according to normal (>3, n 7) or low body condition score (LBCS; <2, n 6), thereby enabling us to examine the effect of maternal body composition on later cardiovascular function. From day 80 to term (approximately 147 d) all sheep were fed to 100 % ME. Offspring were weaned at 12 weeks and pasture-reared until 6 months of age when cardiovascular function was determined. Both LBCS and NR sheep tended to have lower resting systolic (control, 85 (se 2); LBCS, 77 (se 3); NR, 77 (se 3) mmHg) and diastolic blood pressure relative to controls. Total nephron count was markedly lower in both LBCS and NR relative to controls (LBCS, 59 (se 6); NR, 56 (se 12) %). Our data suggest that maternal body composition around conception is as important as the level of nutrient intake during early pregnancy in programming later cardiovascular health.

Detection and regulation of leptin receptor mRNA in ovine mammary epithelial cells during pregnancy and lactation

Adipocyte‐epithelial cell interactions and their secretions are critical determinants of mammary gland development. In this present study, we examined the possible involvement of leptin and its receptors in the process of mammogenesis/lactogenesis. We demonstrated by reverse transcription and polymerase chain reaction analysis that long and short forms of leptin receptors were expressed in the ovine mammary gland during pregnancy and lactation. Furthermore, quantitative determinations, via ribonuclease protection assays, provided evidence that the level of leptin receptor expression was greatest during mid‐pregnancy when active growth of the mammary gland is initiated. Location of the leptin receptors, as determined by in situ hybridization analysis, revealed that leptin receptor transcripts were expressed specifically in mammary epithelial cells. These data provide evidence that leptin, with its receptors, could be an important mediator in regulating mammary gland growth and development.

Central Infusion of Recombinant Ovine Leptin Normalizes Plasma Insulin and Stimulates a Novel Hypersecretion of Luteinizing Hormone after Short-Term Fasting in Mature Beef Cows

Abstract The present studies tested the hypotheses that short-term fasting would reduce leptin gene expression and circulating concentrations of leptin and insulin in mature, ovariectomized, estradiol-implanted cows and that intracerebroventricular infusions of recombinant ovine leptin (oleptin) would attenuate reductions in insulin concentration and stimulate LH secretion. Ovariectomized cows were assigned to either control (normal fed; n = 6) or fasted (60 h of fasting; n = 7) groups and infused with 200 μg recombinant oleptin three times at hourly intervals on Day 2 (n = 6 per group). Fasting decreased plasma concentrations of insulin (P < 0.01) and leptin (P < 0.04) but, as expected, did not reduce plasma concentrations of glucose or any LH secretion variable. Central infusion of leptin on Day 2 increased (P < 0.01) plasma concentrations of leptin in both control and fasted groups. Concomitantly, leptin treatment increased plasma insulin (P < 0.01) and LH (P < 0.03) concentrations in fasted but not in control cows. Increases in overall mean and baseline concentrations of LH after leptin treatment were the result of an augmentation of the size of LH pulses. The effects of fasting on leptin gene expression and the potential diurnal effects on circulating leptin were examined in a group of cows (n = 12) not treated with leptin. Fasting for 60 h reduced (P < 0.001) leptin gene expression by 30%, and no diurnal effects on circulating leptin were observed. These results indicate that although short-term fasting does not reduce the frequency or amplitude of LH pulses or the concentration of LH in mature cows, this nutritional perturbation clearly sensitizes both the hypothalamic-pituitary axis and endocrine pancreas to exogenous leptin, which in these experiments resulted in heightened secretion of both LH and insulin.

Parenteral Administration of l-Arginine Prevents Fetal Growth Restriction in Undernourished Ewes

Intrauterine growth restriction (IUGR) is a major health problem worldwide that currently lacks an effective therapeutic solution. This study was conducted with an ovine IUGR model to test the hypothesis that parenteral administration of l-arginine (Arg) is effective in enhancing fetal growth. Beginning on d 28 of gestation, ewes were fed a diet providing 100% (control-fed) or 50% (underfed) of NRC-recommended nutrient requirements. Between d 60 of gestation and parturition, underfed ewes received i.v. infusions of saline or 155 micromol Arg-HCl/kg body weight 3 times daily, whereas control-fed ewes received only saline. The birth weights of lambs from saline-infused underfed ewes were 23% lower (P < 0.01) than those of lambs from control-fed dams. Administration of Arg to underfed ewes increased (P < 0.01) concentrations of Arg (69%), ornithine (55%), proline (29%), methionine (37%), leucine (36%), isoleucine (35%), cysteine (19%), and FFA (43%) in maternal serum, decreased maternal circulating levels of ammonia (18%) and triglycerides (32%), and enhanced birth weights of lambs by 21% compared with saline-infused underfed ewes. There was no difference in birth weights of lambs between the control-fed and the Arg-infused underfed ewes. These novel results indicate that parenteral administration of Arg to underfed ewes prevented fetal growth restriction and provide support for its clinical use to ameliorate IUGR in humans. The findings also lay a new framework for studying cellular and molecular mechanisms responsible for the beneficial effects of Arg in regulating conceptus growth and development.

Leptin and its role in the central regulation of reproduction in cattle.

Leptin, a 16kDa product of the adipose obese (ob) gene, has been shown to contribute to the regulation of energy metabolism, feeding behavior, and reproduction in several monogastric species, including humans. Recent reports have provided evidence that the leptin gene is functionally relevant in cattle and sheep, and may contribute to an array of important reproductive events, including puberty. Leptin gene expression and circulating leptin increase markedly during sexual maturation in heifers reaching puberty during late spring or early summer. In addition, serum leptin concentrations increased by over 30% from early winter to the summer solstice in mature cows, and also increased with significant changes in adiposity. However, only limited changes in circulating leptin have been observed during the estrous cycle. Short-term fasting of growing peripubertal heifers causes marked reductions in leptin gene expression and circulating leptin, concomitant with declines in LH pulse frequency, and serum concentrations of insulin and IGF-1. Although short-term fasting of mature cows in excellent body condition is without effects on LH pulse frequency, it has remarkably similar metabolic effects to those observed in heifers. Moreover, ICV administration of recombinant oleptin resulted in a marked hypersecretion of LH in fasted cows, and in vitro studies using both hypothalamic and anterior pituitary explants have provided evidence that this effect is at the pituitary level. Paradoxically, ICV administration of oleptin normalized circulating insulin in fasted cows but hleptin was without effect on insulin in estradiol-implanted wethers. Collectively, work in cattle and sheep indicates that leptin can modulate both the hypothalamic-pituitary axis and endocrine pancreas under defined nutritional conditions. Additional work to more fully characterize these roles is clearly warranted and could lead to the development of novel strategies for modifying reproductive potential in food-producing species.

Parenteral Administration of L-Arginine Enhances Fetal Survival and Growth in Sheep Carrying Multiple Fetuses

The frequency of multiple fetuses has increased in human pregnancies due to assisted reproductive technologies. This translates into a greater proportion of premature and low-birth weight infants in the United States and worldwide. In addition, improvements in sheep breeding have resulted in new breeds with increased litter size but reduced fetal survival and birth weight. Currently, there are no treatments for preventing fetal growth restriction in humans or sheep (an established model for studying human fetal physiology) carrying multiple fetuses. In this work, Booroola Rambouillet ewes (FecB+/-) with 2-4 fetuses were fed a diet providing 100% of NRC-recommended nutrient requirements. Between d 100 and 121 of gestation, ewes received an i.v. bolus injection of either saline solution or 345 μmol arginine-HCl/kg body weight 3 times daily. The arginine treatment reduced (P < 0.05) the percentage of lambs born dead by 23% while increasing (P = 0.05) the percentage of lambs born alive by 59%. The i.v. administration of arginine enhanced (P < 0.05) the birth weights of quadruplets by 23% without affecting maternal body weight. The improved pregnancy outcome was associated with an increase in maternal plasma concentrations of arginine, ornithine, cysteine, and proline, as well as a decrease in circulating levels of ammonia and β-hydroxybutyrate. These novel results indicate that parenteral administration of arginine to prolific ewes ameliorated fetal mortality and growth retardation. Our findings provide support for experiments to assess the clinical use of arginine to enhance fetal growth and survival in women gestating multiple fetuses.