Robert R. Kraeling

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.

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.

Developmental Changes in the Long Form Leptin Receptor and Related Neuropeptide Gene Expression in the Pig Brain

Abstract The hypothalamus is the key site of central regulation of energy homeostasis, appetite, and reproduction. The long form leptin receptor (Ob-Rl) is localized within the hypothalamus along with several neuropeptides that are involved in regulation of the neuroendocrine axis. In the present study, developmental changes in gene expression of the Ob-Rl, preproorexin, proopiomelanocortin (POMC), corticotropin releasing factor (CRF), somatostatin, and GnRH in the hypothalamus was studied. Expression of Ob-Rl and neuropeptide mRNA was examined by semiquantitative reverse transcription-polymerase chain reaction in hypothalami collected from 106-day-old fetus (n = 3) and 7-day-old (n = 3), 3.5-mo-old (n = 3), and 6-mo-old (n = 2) gilts. In addition, leptin mRNA expression in the first three ages was examined in back fat. Leptin mRNA expression increased (P < 0.05) by 7 days postnatal, but Ob-Rl mRNA expression increased (P < 0.01) by 3.5 mo. Expression of preproorexin (P < 0.05), somatostatin, and GnRH (P < 0.01) mRNA peaked by 3.5 mo of age while POMC mRNA expression increased markedly (P < 0.01) by 6 mo of age. The CRF mRNA expression did not change across ages. These findings suggest a possible relationship among Ob-Rl and a number of hypothalamic and peripheral peptides in the development of the neuroendocrine axis. These peptides may serve as messengers that link mechanisms that regulate reproduction and energy balance.

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.

Intracerebroventricular porcine corticotropin-releasing hormone and cortisol effects on pig immune measures and behavior

The effects of intracerebroventricular (icv) administration of porcine corticotropin-releasing hormone (pCRH) and cortisol on the immune system and behavior were examined in domestic pigs. In Experiment 1, 50 micrograms of pCRH in 200 microliters of saline or 200 microliters of vehicle was administered i.c.v. at 0600 h. Blood samples were obtained at 0600 (prior to injection), 0700, and 0800 h. Plasma cortisol concentrations were higher at 1 and 2 h after pCRH than after saline. Generally, pCRH failed to effect NK cytotoxicity or lymphocyte proliferation in response to phytohemagluttin (PHA). However, 1 h postinjection, pigs administered pCRH i.c.v. had marginally lower NK activity than control pigs. Pigs injected with pCRH had substantially lower neutrophil chemotaxis (CHTX) than the control pigs at 1 and 2 h postinjection. As blood cortisol concentration increased, neutrophil CHTX decreased. Pigs injected i.c.v. with pCRH had higher neutrophil numbers and neutrophil:lymphocyte ratios than control pigs. Percentage of lymphocytes was higher among control than treated pigs. Central pCRH increased overall activity, particularly walking, standing, licking, rooting, and increased activity-related sequences (e.g., sit, walk and stand, walk), but reduced complex oral/nasal sequences (e.g., root, lick). In Experiment 2, pigs were injected i.c.v. with 10 micrograms of cortisol in 200 microliters of saline or with vehicle at 0600 h. Administration of cortisol failed to effect NK cytotoxicity, lymphocyte proliferation, CHTX, or leukocyte distribution. Pigs given cortisol had no apparent change in behavior. These data indicate leukocyte distribution and specific neutrophil function in pigs were significantly modulated by stress-related hormones of the hypothalamic-pituitary-adrenal axis and complexity of behavioral sequences (pigs repeating certain behavioral sequences) associated with increased activity was reduced. Oral/nasal stereotypies (as seen among confined sows) were not elevated among pigs given i.c.v. pCRH. CRH given by i.c.v. administration may serve as a better model for acute rather than chronic stress.

On the site of action of naloxone-stimulated cortisol secretion in gilts

The increase in serum cortisol concentrations following naloxone administration to female pigs was abolished by hypophysial stalk-transection, even though CRH and ACTH stimulated cortisol release in these animals. We suggest that the opioid antagonist enhances cortisol secretion primarily by a central action in pigs.

Immunocytochemical distribution of catecholamine-synthesizing neurons in the hypothalamus and pituitary gland of pigs: Tyrosine hydroxylase and dopamine-β-hydroxylase

This study describes the distribution of catecholaminergic neurons in the hypothalamus and the pituitary gland of the domestic pig, Sus scrofa, an animal that is widely used as an experimental model of human physiology in addition to its worldwide agricultural importance. Hypothalamic catecholamine neurons were identified by immunocytochemical staining for the presence of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine‐β‐hydroxylase. Tyrosine hydroxylase‐immunoreactive perikarya were observed in the periventricular region throughout the extent of the third ventricle, the anterior and retrochiasmatic divisions of the supraoptic nucleus, the suprachiasmatic nucleus, the ventral and dorsolateral regions of the paraventricular nucleus and adjacent dorsal hypothalamus, the ventrolateral arcuate nucleus, and the posterior hypothalamus. Perikarya ranged from parvicellular (10–15 μm) to magnocellular (25–50 μm) and were of multiple shapes (rounded, fusiform, triangular, or multipolar) and generally had two to five processes with branched arborization. No dopamine‐β‐hydroxylase immunoreactive perikarya were observed within the hypothalamus or in the adjacent basal forebrain structures. Both tyrosine hydroxylase‐ and dopamine‐β‐hydroxylase‐immunoreactive fibers and punctate varicosities were observed throughout areas containing tyrosine hydroxylase perikarya, but dopamine‐β‐hydroxylase immunoreactivity was very sparse within the median eminence. Within the pituitary gland, only tyrosine hydroxylase fibers, and not dopamine‐β‐hydroxylase immunoreactive fibers, were located throughout the neurohypophyseal tract and within the posterior pituitary in both pars intermedia and pars nervosa regions. Generally, the location and patterns of both catecholamine‐synthesizing enzymes were similar to those reported for other mammalian species except for the absence of the A15 dorsal group and the very sparse dopamine‐β‐hydroxylase immunoreactive fibers and varicosities in the median eminence in the pig. These findings provide an initial framework for elucidating behavioral and neuroendocrine species differences with regard to catecholamine neurotransmitters.