Lloyd L. Anderson

Growth hormone secretion: molecular and cellular mechanisms and in vivo approaches.

Growth hormone (GH) release is under the direct control of hypothalamic releasing hormones, some being also produced peripherally. The role of these hypothalamic factors has been understood by in vitro studies together with such in vivo approaches as stalk sectioning. Secretion of GH is stimulated by GH-releasing hormone (GHRH) and ghrelin (acting via the GH secretagogue [GHS] receptor [GHSR]), and inhibited by somatostatin (SRIF). Other peptides/proteins influence GH secretion, at least in some species. The cellular mechanism by which the releasing hormones affect GH secretion from the somatotrope requires specific signal transduction systems (cAMP and/or calcium influx and/or mobilization of intracellular calcium) and/or tyrosine kinase(s) and/or nitric oxide (NO)/cGMP. At the subcellular level, GH release (at least in response to GHS) is accomplished by the following. The GH-containing secretory granules are moved close to the cell surface. There is then transient fusion of the secretory granules with the fusion pores in the multiple secretory pits in the somatotrope cell surface.

Gene expression profiling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed efficiency

Residual feed intake (RFI) is a measure of feed efficiency, in which low RFI denotes improved feed efficiency. Caloric restriction (CR) is associated with feed efficiency in livestock species and to human health benefits, such as longevity and cancer prevention. We have developed pig lines that differ in RFI, and we are interested in identifying the genes and pathways that underlie feed efficiency. Prepubertal Yorkshire gilts with low RFI (n = 10) or high RFI (n = 10) were fed ad libitum or fed at restricted intake of 80% of maintenance energy requirements for 8 days. We measured serum metabolites and hormones and generated transcriptional profiles of liver and subcutaneous adipose tissue on these animals. Overall, 6,114 genes in fat and 305 genes in liver were differentially expressed (DE) in response to CR, and 311 genes in fat and 147 genes in liver were DE due to RFI differences. Pathway analyses of CR-induced DE genes indicated a dramatic switch to a conservation mode of energy usage by down-regulating lipogenesis and steroidogenesis in both liver and fat. Interestingly, CR altered expression of genes in immune and cell cycle/apoptotic pathways in fat, which may explain part of the CR-driven lifespan enhancement. In silico analysis of transcription factors revealed ESR1 as a putative regulator of the adaptive response to CR, as several targets of ESR1 in our DE fat genes were annotated as cell cycle/apoptosis genes. The lipid metabolic pathway was overrepresented by down-regulated genes due to both CR and low RFI. We propose a common energy conservation mechanism, which may be controlled by PPARA, PPARG, and/or CREB in both CR and feed-efficient pigs.

Stimulatory Effect of Ghrelin on Isolated Porcine Somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca2+ concentration – [Ca2+]i – in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca2+]i in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys3)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca2+]i evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca2+]i. The stimulatory effect of ghrelin on somatotropes was greatly attentuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca2+]i transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca2+]i similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca2+]i. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca2+]i. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.

Neonatal Meishan pigs show POU1F1 genotype effects on plasma GH and PRL concentration

Chinese Meishan pigs develop rapidly with onset of puberty at less than 100 days of age, and have a smaller placental size and larger litter size as compared with British/Continental breeds. POU1F1 is a member of the POU-domain family gene and is a positive regulator for growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone beta (TSHbeta) in several mammalian species. To investigate the role of POU1F1 in controlling pig growth and reproduction traits, Meishan (MS) pigs segregating a MspI POU1F1 polymorphism were used to determine differences of GH and PRL at both mRNA and circulating hormone concentrations. Animals from nine litters were used to collect pituitary (n=60) and/or blood samples (n=80) at day 0, 15, and 30 after birth, and all animals were genotyped (CC, CD, DD) for the MspI POU1F1 polymorphism. Reverse transcriptase-polymerase chain reaction (RT-PCR) with standard curve quantification was used to quantify mRNA levels for GH, PRL, and two alternative POU1F1 transcripts, POU1F1-alpha, and POU1F1-beta. Radioimmunoassays were done to determine the circulating concentration of GH and PRL in blood plasma. Our results indicated a significant effect of POU1F1 genotype on circulating levels of both GH and PRL at birth, but not thereafter. The DD neonates had lower levels of GH, but higher levels of PRL, than other genotypes. POU1F1-alpha mRNA decreased (P<0.05) from days 0 to 30, which paralleled decreases (P<0.05) in GH mRNA as well as PRL and GH plasma levels over the same period. POU1F1-beta mRNA levels did not significantly change over this period. Correlations were significant between POU1F1-alpha mRNA and both GH mRNA and GH plasma concentration levels, as well as between the two POU1F1 mRNA isoforms. Results from this study add to our understanding of the role of POU1F1 in controlling pig development and reproduction.

Microarray gene expression profiles of fasting induced changes in liver and adipose tissues of pigs expressing the melanocortin-4 receptor D298N variant

Transcriptional profiling coupled with blood metabolite analyses were used to identify porcine genes and pathways that respond to a fasting treatment or to a D298N missense mutation in the melanocortin-4 receptor (MC4R) gene. Gilts (12 homozygous for D298 and 12 homozygous for N298) were either fed ad libitum or fasted for 3 days. Fasting decreased body weight, backfat, and serum urea concentration and increased serum nonesterified fatty acid. In response to fasting, 7,029 genes in fat and 1,831 genes in liver were differentially expressed (DE). MC4R genotype did not significantly affect gene expression, body weight, backfat depth, or any measured serum metabolite concentration. Pathway analyses of fasting-induced DE genes indicated that lipid and steroid synthesis was downregulated in both liver and fat. Fasting increased expression of genes involved in glucose sparing pathways, such as oxidation of amino acids and fatty acids in liver, and in extracellular matrix pathways, such as cell adhesion and adherens junction in fat. Additionally, we identified DE transcription factors (TF) that regulate many DE genes. This confirms the involvement of TF, such as PPARG, SREBF1, and CEBPA, which are known to regulate the fasting response, and implicates additional TF, such as ESR1. Interestingly, ESR1 controls several fasting induced genes in fat that are involved in cell matrix morphogenesis. Our findings indicate a transcriptional response to fasting in two key metabolic tissues of pigs, which was corroborated by changes in blood metabolites, and the involvement of novel putative transcriptional regulators in the immediate adaptive response to fasting.

Physiological Regulation of Maternal Behavior in Heifers: Roles of Genital Stimulation, Intracerebral Oxytocin Release, and Ovarian Steroids

Abstract We tested the hypotheses that 1) epidural anesthesia at parturition would block both peripheral and central release of oxytocin and eliminate the development of maternal behavior in primiparous heifers and 2) estradiol priming, genital stimulation, and appropriate neonatal stimuli would induce maternal behavior in nulliparous heifers. In experiment 1, primiparous crossbred heifers (n = 13) with cannulas in the third cerebroventricle (IIIV) were assigned randomly to receive epidural treatments of saline (SAL; n = 6) or lidocaine HCl (EPI; n = 7) at the onset of labor induced between Days 270 and 280 of gestation. Epidural anesthesia blocked (P < 0.001) both central and peripheral release of oxytocin and markedly reduced (P < 0.05) or eliminated licking behaviors during a 3-h period following parturition as compared with SAL. Following approximately 1 wk of controlled daily suckling, during which calves were permitted access only to the inguinal region of their dams (three times daily for 10 min each time), a second maternal behavior test was performed. Although licking behavior remained markedly reduced (P < 0.001) in the EPI compared with the SAL groups, all heifers accepted their calf at the udder. In experiments 2–4, neither estradiol priming in ovariectomized heifers nor estradiol plus progesterone in intact heifers resulted in an induction of maternal behaviors following genital stimulation and presentation of a neonate wetted with amniotic fluid. Pelvic sensory deficits apparently block oxytocin release and disturb both short-latency and long-term maternal behaviors but do not result ultimately in rejection of the calf. Combinations of hormonal, sensory, olfactory, and visual cues observed previously to induce maternal behavior in nulliparous ewes do not appear adequate for induction of maternal behavior in nulliparous heifers.

Number of Secretory Vesicles in Growth Hormone Cells of the Pituitary Remains Unchanged After Secretion

Immunogold-labeled transmission electron microscopy (TEM) was used to determine the total number of secretory vesicles in resting and in growth hormone (GH)-stimulated porcine pituitary cells. We identified three categories of vesicles: filled, empty, and partly empty. Resting GH cells contained more than twice as many filled vesicles than did the stimulated ones. Stimulated cells, however, contained nearly twice as many empty vesicles and 2.5 times more partly empty vesicles than did resting cells. Secretory vesicles in GH cells further revealed the localization of GH only in electron-dense vesicles in both resting and stimulated cells. The total number of secretory vesicles did not change after secretion. These results are consistent with a mechanism that, after stimulation of secretion, vesicles transiently dock and fuse at the fusion pore to release vesicular contents.

Growth Hormone and Prolactin Secretion in Hypophysial Stalk-Transected Pigs as Affected by Growth Hormone and Prolactin-Releasing and Inhibiting Factors

Abstract Control of growth hormone (GH) and prolactin (PRL) release was investigated in hypophysial stalk-transected (HST) and stalk-intact pigs by determining the effects of analogs of GH-releasing factors (GHRF), somatostatin (SRIF), arginine, thyrotropin-releasing hormone, α-methyl-ρ-tyrosine, and haloperidol. HST and control gilts were challenged with intravenous injections of human pancreatic GHRF(1–40)OH, thyrotropin-releasing hormone, and analogs of rat hypothalamic GHRF. HST animals remained acutely responsive to GHRF by releasing 2-fold greater quantities of GH than seen in controls. This occurred in spite of a 38% reduction in pituitary gland weight and a 32 and 55% decrease in GH concentration and total content. During SRIF infusion, GH remained at similar basal concentrations in HST and control gilts, but increased immediately after stopping SRIF infusion only in the controls. Releasable pituitary GH appears to accumulate during SRIF infusion. GHRF given during SRIF infusion caused a 2-fold greater release of GH than seen in animals receiving only GHRF. Arginine increased (P < 0.05) GH release in controls, but not in HST gilts, which suggests that it acts through the central nervous system. Basal PRL concentrations were greater (P < 0.05) in HST gilts than in control gilts. TRH acutely elevated circulating PRL (P < 0.001) in HST gilts, suggesting that it acts directly on the pituitary gland. Haloperidol, a dopamine receptor antagonist, increased circulating PRL in controls but not in HST animals. α-Methyl-ρ-tyrosine did not consistently increase circulating PRL, however, suggesting that it did not sufficiently alter turnover rate of the tyrosine hydroxylase pool. The results indicate that the isolated pituitary after HST remains acutely responsive to hypothalamic releasing and inhibiting factors for both GH and PRL release in the pig.

Relaxin-induced expression of Fos in the forebrain of the late pregnant rat.

Relaxin, administered parenterally, has been shown to increase the release of oxytocin (OT) into the circulation and increase the firing rate of OTergic neurons. The objective of the present study was to determine if relaxin administration can result in the expression of a transcription factor, suggesting that it alters transcriptional activity within OTergic neurons at the level of the hypothalamus. Primigravid rats were ovariectomized and a jugular cannula was inserted on day 11 of gestation (g11). Pregnancy was maintained by implanting 17 beta-estradiol and progesterone caplets subcutaneously at the time of ovariectomy. At gl9, rats were challenged with intravenous relaxin or isotonic saline and the brains were removed for study. Immunohistochemistry was performed on coronal brain sections, utilizing Fos as a marker of cellular activation. In the group receiving relaxin, Fos-like immunoreactivity (Fos-IR) was abundant only in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus as well as in the subfornical organ (SFO). In contrast, Fos-IR in the group given isotonic saline was lacking in these three brain regions. A double label study using antibodies against Fos and OT demonstrated that a majority of the Fos-labeled cells in the hypothalamus were OTergic. Because Fos can act as a transcription factor, we interpret these data to indicate that transcription within OTergic cells is altered following relaxin administration, with abundant Fos-IR being limited to the SON and PVN of the hypothalamus and the SFO during late pregnancy in the rat.

Discovery of a new cellular structure--the porosome: elucidation of the molecular mechanism of secretion.

Invention of the light microscope some 300 years ago allowed discovery of the unit of life, the cell. Up until the early 1940s, the light microscope, so efficient in the 19th century, reached the limits of its resolving power, of some 200 nanometer in lateral resolution and much less in its depth resolution. With the invention of the electron microscope and its use in biological research, a new era dawned for cell biology, when major subcellular organelles, and their organization and function, were elucidated. Pioneering cell biologists—George E. Palade, Keith Porter, and Albert Claude—made stellar contributions during this period. With a lateral resolving power of nearer a nanometer, the electron microscope matured into the ultimate imaging tool in the study of the cell. Unlike the light microscope, the electron microscope sacrificed live cell imaging for a considerable gain in resolution. Although, the electron microscope was capable of imaging biological samples at nanometer resolution, it required sample processing that included freezing, fixation, dehydration, and staining with heavy metal compounds to increase contrast. Hence, morphological changes due to freezing, tissue fixation, and processing for electron microscopy have always been a major concern. None the less, using electron microscopy, it appeared we knew just about all there is to know of cellular structure. After nearly half a century, nature has surprised us once more, with the discovery of a new cellular structure, the ‘porosome’, by Bhanu Jena and his research team (Jena et al., 2003; Jeremic et al., 2003; see Figs. 1 and 2). During 1995–96, Jena’s group circumvented the problems faced by both light and electron microscopy in their pioneering studies, by using the Atomic Force Microscope (AFM), which had been developed in the mid 1980s. In a series of elegant studies on live cells, his research team utilized the power and scope of the AFM to discover a new structure at the plasma membrane where membrane-bounded secretory vesicles dock and fuse to release their vesicular contents. The structure was found to be located in the plasma membrane of cells where secretion occurs. Circular ‘pits’ containing 150 nm diameter ‘depressions’ (now identified as porosomes) were present at the apical plasma membrane in living pancreatic acinar cells (Schneider et al., 1997). Stimulation of secretion caused the ‘depressions’ to dilate and then return to their resting size following completion of the process. Exposure of cells to actin depolymerizing agents resulted in collapse of ‘depressions’ and a consequent loss in secretion. Zymogen granules, the membrane-bounded secretory vesicles in exocrine pancreas, contain the starch digesting enzyme amylase. Using amylase-specific immunogold-AFM studies, localization of amylase at ‘depressions’ following stimulation of secretion was demonstrated (Cho et al., 2002c; Jena, 2002). These studies demonstrate that these ‘depressions’ are the fusion pores of pancreatic acinar cells, where membranebound secretory vesicles dock and fuse to release their vesicular contents. Subsequently, a similar presence and operation of fusion pores was confirmed in growth hormone secreting cells of the pituitary gland (Cho et al., 2002a) and in chromaffin cells (Cho et al., 2002e), suggesting their universal presence in secretory cells. Recently, the detailed morphology of the porosome was also revealed using electron microscopy, showing a cup-shaped basket-like morphology (Jena et al., 2003; Jeremic et al., 2003). Using immuno-AFM and biochemical approaches, further studies revealed the composition of the fusion pores, and demonstrated t-SNAREs associated with the base of the cup (Jeremic * Corresponding author. Tel.: +1-515-294-5540; fax: +1-515-294-4471 E-mail address: llanders@iastate.edu (L.L. Anderson). Cell Biology International 28 (2004) 3–5 Cell Biology International