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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Avian Gut and Pancreatic Endocrine Cells: Developmental AspectsB.B. Rawdon Department of Anatomy, University of Cape Town Medical School,
South Africa A similar range of endocrine cell types is found in the gut of birds as in mammals. Endocrine cells first appear in chick gut at 12 days of incubation and all known types are present by hatching. Experimental studies have shown that mesenchyme can profoundly influence the differentiation of avian gut endocrine cells. Although endocrine cell pattern is established early in gut endoderm, it can be modified by exposure of the endoderm to heterologous gut mesenchyme. Recently, we have shown that the specific properties of gut mesenchyme that control endocrine cell differentiation also develop early. Currently we are exploring several lines of investigation aimed at determining the nature of the factors that might influence the "programming" of mesenchyme to support differentiation of gut endocrine cells. The pancreas in birds arises from two ventral and a single dorsal bud and is unusual in that the ratio of insulin to glucagon cells is approximately 1:2, the opposite of the situation in mammals. Endocrine cells can be detected in the dorsal bud on the third day of incubation, well in advance of their differentiation in the ventral buds. We have used the 5day chick dorsal bud as a model system to investigate factors influencing insulin cell development in vitro. The sum of our investigations to date has shown that near normal proportions of insulin cells develop when embryonic explants are grown on a basement membrane extract (Matrigel) in a serumfree medium (Ham's F12) provided the medium is supplemented with insulin, selenium and transferrin and the glucose level raised from 10mM to 20mM. Insulin SecretionN. Rideau Endocrinologie de la Croissance et du Métabolisme, Station de
Recherches Avicoles, INRA, 37380 Nouzilly, France The origin of the hyperglycaemic state (as compared to mammals) and the control of insulin release and its possible implications in the control of growth and body composition are poorly understood in bird species. In contrast to mammals, very high glucose concentrations are required to induce a typical biphasic insulin release from the chicken isolated and perfused pancreas. We have further investigated the stimulus secretion coupling and shown that most of the nutrients which are "primary initiators" of insulin release in mammals: Dglyceraldehyde (515 mM, DGA), Dmannose (2050 mM), Lleucine (2040 mM) and aketoisocaproate (1040 mM, KIC) are either poorly or not efficient at initiating insulin release when perfused alone. An additional but noninsulinotropic fuel supply permits these nutrients to become insulinotropic in the chicken pancreas. Glucose is more efficient than other nutrients (DGA, Lglutamine and Lasparagine) to exert this "permissive" effect. Intracellular metabolism is also required since 3OmethylDglucose does not potentiate the response to KIC. The relative insensitivity of the chicken pancreas can also be overcome by the simultaneous perfusion of cAMP and/or acetylcholine (Ach), according to the nutrient. On the other hand 1040 mM K+ or 20 mM arginine induce a rapid monophasic insulin output. Together, these results suggest that the metabolic threshold which permits the switching of the bcell from the resting to the active state is much higher in chicken than in mammals. By an unknown mechanism, the threshold is lowered more efficiently by glucose than by the other fuel nutrients. The potentiation mechanisms involved may involve Ach or cAMP and involve membrane depolarisation events (K+ or arginine). Studies aimed at understanding the peculiarities of the chicken bcell are presently based on isolated chicken islets of Langerhans. Using this experimental model changes in insulin secretion in response to genetic, nutrional and other physiological factors will subsequently be studied. Insulin Receptors and Insulin SignallingM. Taouis, J. Simon Endocrinologie de la Croissance et du Métabolisme, Station de
Recherches Avicoles, INRA, 37380 Nouzilly, France The insulin receptor contains two _subunits and two ß subunits. Upon insulin binding to the extracellular _subunit, the tyrosine kinase contained in the intracellular part of the ß subunit is activated leading to an autophosphorylation of the ß subunit and to the phosphorylation of one or several cellular substrates. As previously reviewed, insulin sensitivity in chicken tissues is controlled at multiple levels: insulin receptor number and tyrosine kinase activity and most likely at one or several steps of the cascade activated by the receptor kinase. In mammals, one major insulin receptor substrate (IRS1) has been identified as a docking protein which, following tyrosine phosphorylation, is able to interact with several intracellular proteins which, in a cascade, activate most of, if not all, the metabolic and mitogenic pathways controlled by insulin. Using chicken genomic DNA, the chicken IRS1 gene has been cloned and the coding region sequenced. It appears to be highly conserved (Taouis et al., submitted). The protein sequence of chicken IRS1, ignoring gaps, is 91 % identical to the human homologue. Most of the tyrosine sites which are important for interaction with proteins containing SH2 regions are present at about the same positions; the pleckstrin region is also highly conserved. The IRS1 mRNA is present very early in chicken embryos and following hatching, in the main target tissues (liver, muscle and adipose tissue). Furthermore, IRS1 mRNA is also expressed in a chicken hepatoma cell line (LMH) and in those cells, the IRS1 protein exhibit an insulin dependent phosphorylation of tyrosine residues. It is therefore very like that in the chicken, IRS1 also serves as a key step in the control of insulin and IGF signalling (IRS1 is also a substrate for IGF1 receptor). Multifactorial Hypothalamic Control of the Thyroid Axis during Growth and Development of the ChickenE.R. Kühn, K.L. Geris, L.R. Berghman, J. Buyse, V.M. Darras Leuven Poultry Research Group, Naamsestraat 61, B-3000 Leuven,
Belgium In the chick embryo injections of TRH increase plasma concentrations of both TSH and GH. TSH is purely thyrotropic and mainly elevates plasma concentrations of T4, whereas GH inhibits hepatic type III deiodination (IRD-III) resulting in increased plasma concentrations of T3. In the early juvenile chicken the thyrotropic effect of TSH remains, but GH or TRH-or GRH-induced GH release is ineffective in increasing plasma concentrations of T3, due to an already existing maximal inhibitory effect on hepatic IRD-III. This may explain why injections of GH or T3 are ineffective in stimulating growth in the chicken and it is challenging to suggest that any conditions which increase IRD-III (hypophysectomy, starvation) may facilitate a GH and consequently T3 mediated growth stimulation. CRH increases thyroid hormone secretion in the chick embryo in vitro and in vivo by releasing significant amounts of TSH. Simultaneously ACTH and corticosterone are released, which block (as does GH) hepatic IRD-III and increase T3 without affecting T4. An injection of CRH in the posthatch chicken also has an acute thyrotropic effect through its proposed TSH releasing activity. It appears, however, to have a latent secondary effect via ACTH, which increases hepatic IRD-III and decreases plasma T3 levels, opposite to that in embryos. In conclusion, the thyrotropic, somatotropic and corticotropic axis seem to work together to increase the availability of active T3 in the chick embryo, whereas in the posthatch chicken the corticotropic axis may respond to stress by reducing this availability as an energy conservation measure. Hormonal and Nutritional Influence on Thyroid Hormone Deiodinating EnzymesV.M. Darras, J. Buyse, E. Decuypere, E.R. Kühn Leuven Poultry Research Group, Naamsestraat 61, B-3000 Leuven,
Belgium The chicken liver is a very important organ for peripheral deiodination of T4 and contains two deiodinating enzymes: a type I enzyme with both outer ring deiodinating (ORD) and inner ring deiodinating (IRD) activity, and a type III enzyme with only IRD activity. Their amount can be measured with specific in vitro tests under saturating conditions (ORD-I and IRD-III). Several pituitary hormones acutely and independently influence the amount of both enzymes and their effect may vary depending on the ontogenic stage of the chicken. Chicken GH does not affect ORD-I but decreases IRD-III within 2 h after injection, corresponding in vivo with an increase in plasma T3. This effect is best observed in embryos, where endogenous GH levels are low. Chicken PRL injection acutely decreases plasma T4. This is accompanied by increased IRD-III in both embryos and posthatch chicks, while ORD-I is decreased only in posthatch animals. Injections of glucocorticoids in chicken embryos clearly increase plasma T3 and decrease plasma T4. They decrease IRD-III within 1 h and increase ORD-I after 24 hrs. In posthatch chicks the effect on plasma T3 is greatly diminished, as well as the effect on IRD-III, but ORD-I decreases within 1 h after injection. Submitting juvenile chickens to a 4-week period of restricted food intake results in increased plasma T4 and decreased plasma T3 levels. The same effect is found after 1-3 days of complete fasting. In both situations IRD-III is strongly increased while ORD-I is unchanged. When fasted chickens are re-fed, IRD-III starts to decrease within 15 min and returns to normal levels within 2 h. Plasma T3 increases from after 1.5 h of the onset of feeding. In conclusion, both hormonal and nutritional factors can regulate plasma T3 levels by changing the amount of hepatic ORD-I and/or IRD-III and thereby interfering with the balance between T3 producing and T3 degrading activity. Insulin Release by Dorsal and Ventral Islets of Langerhans Isolated from the Chicken PancreasL. Ruffier, S. Crochet, N. Rideau Endocrinologie de la Croissance et du Metabolisme, Station de
Recherches Avicoles, INRA, 37380 Nouzilly, France A technique to isolate islets of Langerhans from the chicken pancreas has been developed in order to understand the chicken b cell insensitivity to "insulinotropic" fuel nutrients (Rideau, Comp. Biochem. Physiol. 103A, 739, 1992). Since the chicken pancreas contains two types of islets (a and b), islets were first isolated from the dorsal and ventral lobes of the chicken pancreas i.e. mainly b islets, producing insulin (Weir et al. Diabetologia 12: 129, 1976). Male broiler chickens (4 to 6 weeks old) were anaesthetized with pentothal and decapitated; the pancreas was rapidly removed, the dorsal and ventral lobes were dissected, minced with scissors and digested during 30 min at 38°C in a Hanks buffer with collagenase (1mg/ml) and BSA (1%). After washing the digest, islets (60 130 mm) were handpicked under a stereomicroscope. Islets appeared as blue irregular ovoid masses. Insulin content was related to the chicken body weight and/or age, with no difference between dorsal or ventral lobes (respectively 2.3±0.3, n=62 and 2.2±0.2, n=70, ng insulin/ml). Replicates of eight islets were incubated during 90 min in a KrebsRinger bicarbonate oxygenated buffer with BSA (5mg/ml) at 38°C in a gently shaking water bath. Different concentrations of glucose (2.8 100 mM) were compared. Insulin release was significantly and repeatedly reduced (29%) but insulin content of the islets was not significantly modified when the islets were exposed to 42 or 100 mM glucose. On the other hand, the presence of 14 mM glucose +1mM acetylcholine did not enhance insulin release. The repeatable and significant inhibition of insulin release in response to high glucose concentrations suggests that the islets are reactive, but (for some unknown reason) they do not express an insulinotropic response to glucose. It is possible that isolated chicken b islets behave as purified rat b cells, which are also unsensitive to glucose (Pipeleers et al. Endocrinology 17: 824, 1985). Isolation and Characterisation of Muscovy (Cairina moschata) Duck InsulinB. Chevalier, P. Anglade1, M. Derouet, D. Mollé2, J. Simon Endocrinologie de la Croissance et du Métabolisme, Station de
Recherches Avicoles, INRA, 37380 Nouzilly; 1Unité Protéines,
Station de Recherches Laitières, INRA, 78352 Jouy-en-Josas;
2Laboratoire de Recherche de Technologie Laitière, INRA, 65 Rue
de Saint Brieuc, 35042 Rennes, France Ducks (Anatidae Family, Anseriform order) are divided in two genera : Peking duck (Anas platyrhynchos genus) and Muscovy duck (Cairina moschata genus). These species differ in their number of liver insulin receptors (despite rather similar plasma insulin levels). The possibility that the presence of different endogenous insulins accounts for the difference in insulin receptor number led us to purify, sequence and characterise the binding properties of Muscovy duck insulin. The sequence of Muscovy duck insulin (measured molar mass: 5729.11) was identical to that described in two other species from the Anseriforme order: Peking duck and goose. The binding affinity of Muscovy duck insulin for rat liver insulin receptors (either membrane bound or solubilized receptors) was much lower than that of porcine insulin (0.3), which most likely accounts for the low biological potency of Peking duck insulin previously described. In contrast, liver receptors from chicken and both duck species exhibited the same affinity for duck and porcine insulin, suggesting the presence of specific structural changes in bird liver insulin receptors. The decrease in the number of insulin receptors in Muscovy duck liver is not therefore a consequence of a change in the insulin molecule. In addition, a comparison between reptilian and avian insulins suggests that the hypoactive "duck type" insulin appeared during the evolution of Aves after the evolution of the hyperactive "chicken type" insulin. Factors Involved in the Differentiation of Chick Insulin Cells in CultureA. Andrew, B.B. Rawdon1 University of the Witwatersrand Medical School, Johannesburg;
1Department of Anatomy, University of Cape Town Medical School,
South Africa Insulin cells are virtually absent in the embryonic chick
pancreas cultured in collagen gel in Basal Medium Eagle,
containing foetal calf serum (BME). To determine factors involved
in the differentiation of chick insulin cells in culture we
substituted Matrigel for collagen gel, and used a richer
serum-free medium, Ham's F12, with insulin, transferrin and
selenium (F12.ITS). Dorsal pancreatic buds of 5d chick embryos
were stripped of enveloping mesenchyme and cultured on Matrigel
for 7d in F12. ITS or in F12.ITS with reduced growth factors
(RGF) or media supplements. Cultures were freeze-dried,
vapour-fixed and embedded in resin, and one-micron sections
immunostained for insulin and glucagon. Counts of these cells
were expressed as a percentage of insulin plus glucagon cell
numbers. Molecular Cloning of the Chicken Type I and III Iodothyronine DeiodinasesS. Van der Geyten1, J.P. Sanders, V.M. Darras1, E.R. Kühn1, J.L. Leonard2, T.J. Visser Department of Internal Medicine III, Erasmus University
Medical School, Rotterdam, The Netherlands; 1Zoological
Institute, Catholic University of Leuven, Leuven, Belgium;
2Department of Nuclear Medicine, University of Massachusetts,
Worcester, MA, USA In embryonic chicken liver (ECL) two types of iodothyronine deiodinases (IDs) are expressed, e.g. ID-I and ID-III. ID-I is able to activate T4 by outer ring deiodination (ORD) to T3 as well as to inactivate T4 by inner ring deiodination (IRD) to rT3, and is predominantly expressed during the later stages of embryonic development. ID-III only catalyzes IRD of T4 and T3, and shows peak expression at earlier stages of embryonic development. Based on two conserved ID aminoacid sequences (NFGSCT and YIEEAH), oligonucleotides were synthesized and used in RT-PCR on poly-A+ RNA isolated from day 17 ECL. This resulted in the amplification of two different DNA fragments, both 117 bp in length. These DNA fragments were used to screen a cDNA library prepared from day 17 ECL poly-A+ RNA. Two clones were isolated, each corresponding to one of the RT-PCR fragments. Based on sequence homology, one clone (1600 bp) was identified as coding for ID-I, while the other clone (2300 bp) corresponds to ID-III. Sequence analysis also revealed that both enzymes contain a selenocystine residue, which is thought to be the catalytic center of these enzymes. The availability of these cDNA clones allows the investigation of the regulation of the mRNA expression of ID-I and ID-III in the developing chicken. Thyroid Function in OstrichesA. Dawson Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton,
Huntingdon, Cambs. PE17 2LS UK Thyroid function in ostriches is of interest because, like other ratites, ostriches are thought to be neotenous descendants of flying birds, and, in amphibia, neoteny is associated with thyroid function. We measured plasma thyroxine in farmed ostriches; juveniles at 5 and 10 months old, and adults (more than 3yr). Mean values were 3.1, 3.2 and 1.8nmol.l-1 respectively (not significantly different). The range of values was striking (e.g. 0.2 -6.5nmol.l-1; CV 90% in adults). The mean values were much lower, and the ranges much greater, than in other avian species. In another group of birds, sampled from hatch until 13 weeks old, mean thyroxine decreased from 7.6 to 2.3nmol.l-1 by 10 days and then remained below 2nmol.l-1. Again values varied widely, but varied within individuals with time rather than individuals being consistently different. We also compared thyroid function in ostriches with quail and starlings. In all three, treatment with TRH (3µg/kg) decreased thyroxine slightly in excess of the decrease caused by repeated sampling. TSH (0.1IU/kg) resulted in a highly significant increase in starlings and quail, but not in ostriches, where it only compensated for the stress induced decrease. The results suggest that the thyroid of ostriches may function more autonomously than in other birds. Peripheral Thyroxine 5'Monodeiodination and Rhythm in Avian Thyroid FunctionP. Prakash, a. Kar1, P. Kumar2, M. Laloraya2, M.S. Parihar3 Govt. College ChachauraBinaganj, Guna (M.P.) India; 1School of
Life Sciences, Indore University (M.P.); 2The Population Council,
Centre for Biomedical Research 1230 York Avenue, NY 10021, USA;
3School of Studies in Zoology, Vikram University, Ujjain (M.P.)
456010, India The Indian rock pigeon, Columba livia intermedia
exhibits circannual and circadian variations in serum thyroid
hormone (T4, T3) concentrations, T3/T4 ratio and tissue (liver
and kidney) type I thyroxine 5' monodeiodinase (5'D) activity. In
this study: Absolute Quantification of MRNA for the Nuclear T3 Receptor in Liver of Broilers of 2 Genetic Lines, by Competitive RT-PCRN. Buys, G. Rahimi, G. Volckaert, E. Decuypere Leuven Poultry Research Group Kardinaal Mercierlaan 92 blok E,
B-3001 Heverlee, Belgium The c-erbA gene codes for a nuclear T3 receptor which mediates multiple effects of T3 that are essential for an adequate metabolism in the chicken. Little is known however about the regulation of this receptor at the gene level. We have developed a quantitative RT-PCR assay to quantify the c-erbA mRNA in liver tissue, which is based on the co-amplification of an in vitro generated transcript differing in one restriction site from the wild type c-erbA mRNA. The pF1_ plasmid, containing the c-erbA sequence was mutated by ligating an EcoRI restriction site into the original EcoRV restriction site and an in vitro transcript was generated. Total RNA was isolated from liver tissue of 10 birds from 2 genetic stocks: one selected for a favourable FCR (FC) and the other one for fast growth (GL). Identical amounts (1µg) of a total RNA sample were spiked with different, two-fold, dilutions (from 40 pg to 2.5 pg) of internal standard RNA, converted to cDNA and amplified by PCR using specific primers. The ratio of co-amplified DNA derived from standard RNA was determined after restriction digestion with EcoRV and separation by agarose gel electrophoresis. This resulted in a series of double bands in which the upper band (144 bp) originates from mutant RNA and the lower one (66bp + 67bp) originates from c-erbA RNA from tissue. The relative amount of each band was measured by densitometry. For each RNA sample a standard curve was plotted and the initial amount of c-erbA mRNA in tissue was calculated from the 50 % level. The procedure was repeated for all liver samples. No line differences in c-erbA gene expression were observed (GL line: 6.2 ± 1.6 pg c-erbA mRNA/µg total RNA and FC line: 6.7 ± 1.5 pg c-erbA mRNA/µg total RNA). |