C.R. Barb

Recombinant Porcine Leptin Reduces Feed Intake and Stimulates Growth Hormone Secretion in Swine

Two experiments (EXP) were conducted to test the hypothesis that porcine leptin affects GH, insulin-like growth factor-I (IGF-I), insulin, thyroxine (T4) secretion, and feed intake. In EXP I, prepuberal gilts received intracerebroventricular (i.c.v.) leptin injections. Blood was collected every 15 min for 4 hr before and 3 hr after i.c.v. injections of 0.9% saline (S; n = 3), 10 micrograms (n = 4), 50 micrograms (n = 4), or 100 micrograms (n = 4) of leptin in S. Pigs were fed each day at 0800 and 1700 hr over a 2-wk period before the EXP. On the day of the EXP, pigs were fed at 0800 hr and blood sampling started at 0900 h. After the last sample was collected, feeders were placed in all pens. Feed intake was monitored at 4, 20, and 44 hr after feed presentation. In EXP II, pituitary cells from prepuberal gilts were studied in primary culture to determine if leptin affects GH secretion at the level of the pituitary. On Day 4 of culture, 10(5) cells/well were challenged with 10(-12), 10(-10), 10(-8), or 10(-6) M [Ala15]-h growth hormone-releasing factor-(1-29)NH2 (GRF), 10(-14), 10(-13), 10(-12), 10(-11), 10(-10), 10(-9), 10(-8), 10(-7), or 10(-6) M leptin individually or in combinations with 10(-8) and 10(-6) M GRF. Secreted GH was measured at 4 hr after treatment. In EXP I, before injection, serum GH concentrations were similar. Serum GH concentrations increased (P < 0.01) after injection of 10 micrograms (21 +/- 1 ng/ml), 50 micrograms (9 +/- 1 ng/ml), and 100 micrograms (13 +/- 1 ng/ml) of leptin compared with S (1 +/- 2 ng/ml) treated pigs. The GH response to leptin was greater (P < 0.001) in 10 micrograms than 50 or 100 micrograms leptin-treated pigs. By 20 hr the 10, 50, and 100 micrograms doses of leptin reduced feed intake by 53% (P < 0.08), 76%, and 90% (P < 0.05), respectively, compared with S pigs. Serum IGF-1, insulin, T4, glucose, and free fatty acids were unaffected by leptin treatment. In EXP II, relative to control (31 +/- 2 ng/well), 10(-10), 10(-8), and 10(-6) M GRF increased (P < 0.01) GH secretion by 131%, 156%, and 170%, respectively. Only 10(-6) M and 10(-7) M leptin increased (P < 0.01) GH secretion. Addition of 10(-11) and 10(-9) M leptin in combination with 10(-6) M GRF or 10(-11) M leptin in combination with 10(-8) M GRF-suppressed (P < 0.05) GH secretion. These results indicate that leptin modulates GH secretion and, as shown in other species, leptin suppressed feed intake in the pig.

Long form leptin receptor mRNA expression in the brain, pituitary, and other tissues in the pig.

Much effort has focused recently on understanding the role of leptin, the obese gene product secreted by adipocytes, in regulating growth and reproduction in rodents, humans and domestic animals. We previously demonstrated that leptin inhibited feed intake and stimulated growth hormone (GH) and luteinizing hormone (LH) secretion in the pig. This study was conducted to determine the location of long form leptin receptor (Ob-Rl) mRNA in various tissues of the pig. The leptin receptor has several splice variants in the human and mouse, but Ob-Rl is the major form capable of signal transduction. The Ob-Rl is expressed primarily in the hypothalamus of the human and rodents, but has been located in other tissues as well. In the present study, a partial porcine Ob-Rl cDNA, cloned in our laboratory and specific to the intracellular domain, was used to evaluate the Ob-Rl mRNA expression by RT-PCR in the brain and other tissues in three 105 d-old prepuberal gilts and in a 50 d-old fetus. In 105 d-old gilts, Ob-Rl mRNA was expressed in the hypothalamus, cerebral cortex, amygdala, thalamus, cerebellum, area postrema and anterior pituitary. In addition, Ob-Rl mRNA was expressed in ovary, uterine body, liver, kidney, pancreas, adrenal gland, heart, spleen, lung, intestine, bone marrow, muscle and adipose tissue. However, expression was absent in the thyroid, thymus, superior vena cava, aorta, spinal cord, uterine horn and oviduct. In the 50 d-old fetus, Ob-Rl mRNA was expressed in brain, intestine, muscle, fat, heart, liver and umbilical cord. These results support the idea that leptin might play a role in regulating numerous physiological functions.

Biology of leptin in the pig

The recently discovered protein, leptin, which is secreted by fat cells in response to changes in body weight or energy, has been implicated in regulation of feed intake, energy expenditure and the neuroendocrine axis in rodents and humans. Leptin was first identified as the gene product found deficient in the obese ob/ob mouse. Administration of leptin to ob/ob mice led to improved reproduction as well as reduced feed intake and weight loss. The porcine leptin receptor has been cloned and is a member of the class 1 cytokine family of receptors. Leptin has been implicated in the regulation of immune function and the anorexia associated with disease. The leptin receptor is localized in the brain and pituitary of the pig. The leptin response to acute inflammation is uncoupled from anorexia and is differentially regulated among swine genotypes. In vitro studies demonstrated that the leptin gene is expressed by porcine preadipocytes and leptin gene expression is highly dependent on dexamethasone induced preadipocyte differentiation. Hormonally driven preadipocyte recruitment and subsequent fat cell size may regulate leptin gene expression in the pig. Expression of CCAAT-enhancer binding proteinalpha (C/EBPalpha) mediates insulin dependent preadipocyte leptin gene expression during lipid accretion. In contrast, insulin independent leptin gene expression may be maintained by C/EBPalpha auto-activation and phosphorylation/dephosphorylation. Adipogenic hormones may increase adipose tissue leptin gene expression in the fetus indirectly by inducing preadipocyte recruitment and subsequent differentiation. Central administration of leptin to pigs suppressed feed intake and stimulated growth hormone (GH) secretion. Serum leptin concentrations increased with age and estradiol-induced leptin mRNA expression in fat was age and weight dependent in prepuberal gilts. This occurred at the time of expected puberty in intact contemporaries and was associated with greater LH secretion. Further work demonstrated that leptin acts directly on pituitary cells to enhance LH and GH secretion, and brain tissue to stimulate gonadotropin releasing hormone secretion. Thus, development of nutritional schemes and (or) gene therapy to manipulate leptin secretion will lead to practical methods of controlling appetite, growth and reproduction in farm animals, thereby increasing efficiency of lean meat production.

Concentrations of leptin in serum and milk collected from lactating sows differing in body condition

Leptin concentrations in the circulation and milk were determined in sows that differed in body condition at farrowing, and in feed consumption during lactation. Serum concentrations of leptin at farrowing and weaning were highest in sows exhibiting the greatest amount of backfat. Leptin was detected in both skim and whole milk throughout lactation, but levels were not correlated with backfat thickness or circulating leptin concentrations. This report provides the first evidence for the presence of leptin in sow milk; its function in the physiology of suckling pigs remains to be determined.

Leptin mRNA expression and serum leptin concentrations as influenced by age, weight, and estradiol in pigs.

Two experiments (EXP) were conducted to determine the roles of age, weight and estradiol (E) treatment on serum leptin concentrations and leptin gene expression. In EXP I, jugular blood samples were collected from gilts at 42 to 49 (n = 8), 105 to 112 (n = 8) and 140 to 154 (n = 8) d of age. Serum leptin concentrations increased (P < 0.05) with age and averaged 0.66, 2.7, and 3.0 ng/ml (pooled SE 0.21) for the 42- to 49-, 105- to 112-, and 140- to 154-d-old gilts, respectively. In EXP II, RNase protection assays were used to assess leptin mRNA in adipose tissue of ovariectomized gilts at 90 (n = 12), 150 (n = 11) or 210 (n = 12) d of age. Six pigs from each age group received estradiol (E) osmotic pump implants and the remaining animals received vehicle control implants (C; Day 0). On Day 7, back fat and blood samples were collected. Estradiol treatment resulted in greater (P < 0.05) serum E levels in E (9 +/- 1 pg/ml) than C (3 +/- 1 pg/ml) pigs. Serum leptin concentrations were not affected by age, nor E treatment. Leptin mRNA expression was not increased by age in C pigs nor by F in 90- and 150-d-old pigs. However, by 210 d of age, leptin mRNA expression was 2.5-fold greater (P < 0.01) in E-treated pigs compared to C animals. Serum insulin concentrations were similar between treatments for 210-d-old pigs. However, insulin concentrations were greater (P < 0.05) in E than C pigs at 90 d and greater in C than E animals at 150 d. Plasma glucose and serum insulin-like growth factor-I concentrations were not influenced by treatment. These results demonstrate that serum leptin concentrations increased with age and E-induced leptin mRNA expression is age- and weight-dependent.

Serum leptin concentrations, luteinizing hormone and growth hormone secretion during feed and metabolic fuel restriction in the prepuberal gilt

Two experiments were conducted to determine 1) the effect of acute feed deprivation on leptin secretion and 2) if the effect of metabolic fuel restriction on LH and GH secretion is associated with changes in serum leptin concentrations. Experiment (EXP) I, seven crossbred prepuberal gilts, 66 +/- 1 kg body weight (BW) and 130 d of age were used. All pigs were fed ad libitum. On the day of the EXP, feed was removed from four of the pigs at 0800 (time = 0) and pigs remained without feed for 28 hr. Blood samples were collected every 10 min from zero to 4 hr = Period (P) 1, 12 to 16 hr = P 2, and 24 to 28 hr = P 3 after feed removal. At hr 28 fasted animals were presented with feed and blood samples collected for an additional 2 hr = P 4. EXP II, gilts, averaging 140 d of age (n = 15) and which had been ovariectomized, were individually penned in an environmentally controlled building and exposed to a constant ambient temperature of 22 C and 12:12 hr light: dark photoperiod. Pigs were fed daily at 0700 hr. Gilts were randomly assigned to the following treatments: saline (S, n = 7), 100 (n = 4), or 300 (n = 4) mg/kg BW of 2-deoxy-D-glucose (2DG), a competitive inhibitor of glycolysis, in saline iv. Blood samples were collected every 15 min for 2 hr before and 5 hr after treatment. Blood samples from EXP I and II were assayed for LH, GH and leptin by RIA. Selected samples were quantified for glucose, insulin and free fatty acids (FFA). In EXP I, fasting reduced (P < 0.04) leptin pulse frequency by P 3. Plasma glucose concentrations were reduced (P < 0.02) throughout the fast compared to fed animals, where as serum insulin concentrations did not decrease (P < 0.02) until P 3. Serum FFA concentrations increased (P < 0.02) by P 2 and remained elevated. Subcutaneous back fat thickness was similar among pigs. Serum IGF-I concentration decreased (P < 0.01) by P 2 in fasted animals compared to fed animals and remained lower through periods 3 and 4. Serum LH and GH concentrations were not effected by fast. Realimentation resulted in a marked increase in serum glucose (P < 0.02), insulin (P < 0.02), serum GH (P < 0.01) concentrations and leptin pulse frequency (P < 0.01). EXP II treatment did not alter serum insulin levels but increased (P < 0.01) plasma glucose concentrations in the 300 mg 2DG group. Serum leptin concentrations were 4.0 +/- 0.1, 2.8 +/- 0.2, and 4.9 +/- 0.2 ng/ml for S, 100 and 300 mg 2DG pigs respectively, prior to treatment and remained unchanged following treatment. Serum IGF-I concentrations were not effected by treatment. The 300 mg dose of 2DG increased (P < 0.0001) mean GH concentrations (2.0 +/- 0.2 ng/ml) compared to S (0.8 +/- 0.2 ng/ml) and 100 mg 2DG (0.7 +/- 0.2 ng/ml). Frequency and amplitude of GH pulses were unaffected. However, number of LH pulses/5 hr were decreased (P < 0.01) by the 300 mg dose of 2DG (1.8 +/- 0.5) compared to S (4.0 +/- 0.4) and the 100 mg dose of 2DG (4.5 +/- 0.5). Mean serum LH concentrations and amplitude of LH pulses were unaffected. These results suggest that acute effects of energy deprivation on LH and GH secretion are independent of changes in serum leptin concentrations.

Growth Hormone-Releasing Hormone and Somatostatin Neurons within the Porcine and Bovine Hypothalamus

Hypothalamic growth hormone-releasing hormone (GHRH) and somatotropin release-inhibiting factor or somatostatin (SS) immunoreactive (ir) neurons were localized in pigs (n = 8) and cattle (n = 7) to identify neuroanatomical sites involved in the regulation of growth hormone secretion. Coronal and sagittal frozen sections (30-60 microns) of Zambonis fixed hypothalamic tissue, without prior colchicine treatment were incubated with GHRH or SS primary antisera for 48 h, then visualized by peroxidase-diaminobenzidine immunocytochemistry. Fusiform, bipolar SS-ir perikarya were located about the third ventricle in the periventricular nucleus, extending from rostral aspects of preoptic periventricular nucleus to a level approximate with caudal regions of the paraventricular nucleus. Rounded or fusiform, bipolar GHRH-ir perikarya were mostly located in ventrolateral portions of the arcuate nucleus in pigs and cattle, and within ventral aspects of the ventromedial nucleus in pigs but rarely in cattle. In both pigs and cattle, SS-ir and GHRH-ir fibers projected ventrally into the median eminence with dense and overlapping innervation of the external layer, especially dense in lateral regions. In pigs, but not as distinguishable in cattle, SS-ir fibers also densely innervated the ventromedial and arcuate hypothalamic nuclei. Double immunostained sections revealed close apposition of SS-ir fibers and varicosities with GHRH-ir perikarya in arcuate and ventromedial nuclei, and apposition of SS-ir and GHRH-ir varicosities in the median eminence.

Opioid modulation of gonadotropin and prolactin secretion in domestic farm animals

Endogenous opioid peptides (EOP) are each derived from one of three distinct precursor molecules: proopiomelanocort in (POMC), proenkephalin and prodynorphin. The POMC molecule gives rise to [3-endorphin while methionine-enkephalin and leucine-enkephalin are pentapeptides produced from the proenkephalin precursor molecule. Finally, the prodynorphin precursor molecule gives rise to ~xand 13-neo-endorphins, as well as dynorphin A and dynorphin B. These products are found in loci throughout the brain and in the pituitary gland. It is not the intention of this review to discuss the biochemistry of the EOP as several reviews have been published addressing this area (1,2,3). At least three major subtypes of EOP receptors appear to exist, namely the m#.-, 8and K-opioid receptors. The classification of receptors is based upon relative affinities and bioassay potencies of different EOP agonists and antagonists (4). Although EOP interact with all types of opioid receptors, provided their concentrations are high enough, products of the prodynorphin and proenkephalin precursors are generally associated with K and B receptors, respectively, while f~-endorphin preferentially binds 8 and mp, receptors (4). Anatomical evidence from immunocytochemical studies in the pig (5), sheep (6), and cow (7), indicate that POMC-immunoreactive perikarya are located within the arcuate area of the hypotha lamus while POMC-immunoreact ive fibers are found in the median eminence.

Aspartate and glutamate modulation of growth hormone secretion in the pig: Possible site of action

The influence of excitatory amino acids (EAA) on growth hormone (GH) secretion and the possible site of action was investigated in the pig. In Experiment (Exp) I three replicates were conducted with 30 prepuberal gilts, 130 d of age and averaging 70.6 +/- 1.3 kg body weight (BW). Six gilts each received intravenously (i.v.) 0, 50, 100, or 150 mg/kg BW of aspartate (ASP) or glutamate (GLU) in saline. Blood samples were collected every 15 min for 2 hr before and 3 hr after treatment. In Exp II, mature ovariectomized gilts (163 +/- 10 kg BW) that had been immunized against growth hormone releasing factor (GRF) conjugated to human serum albumin (GRFi; n = 4) or against human serum albumin alone (HSAi; n = 5) received 150 mg/kg BW ASP or GLU i.v. in a 2 x 2 factorial arrangement of treatments, which was then repeated in a crossover design. One week later, all animals received 10 mg/kg N-methyl-D,L-aspartate (NMA; EAA agonist) in saline i.v. Blood samples were collected as described above. In Exp III, cultures of anterior pituitary cells from market-weight (averaging 105 kg BW) gilts were studied. On Day 4 of culture, cells (10(5) seeded/well) were challenged with 10(-8), 10(-6), or 10(-4) M ASP or GLU, 10(-6) M [Ala15]-human GRF (1-29)-NH2, or the EAA antagonist, 2-amino-5-phosphonopentanoic acid (10(-4) M; AP5), alone or in combination with ASP or GLU. In Exp I, all doses of ASP and the 100- and 150-mg doses of GLU increased (P < 0.05) GH secretion when compared with Time 0. However, serum GH concentrations were higher (P < 0.01) after 150 mg/kg of ASP when compared with those after 150 mg/kg of GLU. In Exp II, serum GH concentrations increased (P < 0.05) in HSAi but not in GRFi pigs (averaging 1.2 +/- 0.2 ng/ml before and 8.2 +/- 0.7, 6.3 +/- 0.5, and 9.2 +/- 0.5 ng/ml by 15 min after ASP, GLU, and NMA, respectively). In Exp III, relative to controls (40 +/- 6 ng/ml), GH increased (P < 0.05)1.6-, 1.9-, and 1.9-fold and 1.7-, 1.8-, and 2.0-fold after 10(-8), 10(-6), and 10(-4) M ASP and GLU, respectively. The EAA receptor antagonist AP5 failed to prevent the GH response to ASP or GLU, except for 10(-8) M ASP. In summary, ASP is a more potent secretagogue of GH secretion than is GLU in vivo, whereas each is equipotent in vitro. Because no stimulation of GH by EAA was observed in GRFi pigs and no specific dose-response effect of EAA was found in vitro, it may be concluded that modulation by EAA is mediated primarily at the level of the hypothalamus or higher brain centers.

Naloxone infusion increases pulsatile luteinizing hormone release in postpartum beef cows

The limiting factor in the return to cyclicity in the postpartum cow appears to be the lack of pulsatile luteinizing hormone (LH) secretion. To test the role of endogenous opioids in regulating pulsatile LH release, naloxone, an opioid antagonist, was infused into postpartum cows. Eight cows (39.3 ± 2.1 d postpartum) received either a constant infusion of saline or 50 mg/hr of naloxone dissolved in saline for eight hr. Blood samples were taken at 15 min intervals for determination of serum LH concentrations and to determine frequency and amplitude of the LH pulses. Frequency of LH pulses was greater (P .05). Mean serum LH concentrations were greater (P<.01) in the naloxone infused group (2.7 ± .3 ng/ml) compared to the saline infused group (1.9 ± .4 ng/ml). These data indicate that endogenous opioids inhibit pulsatile LH secretion in the anestrous postpartum beef cow and that naloxone infusion increases mean serum LH concentrations by increasing the frequency of LH pulses.