Camp. Biochem. Ph@ol. Vol. 96A, No. 2, pp. 327-331, 1990
0300-9629/90$3.00+ 0.00 iQ 1990Pergamon Press plc
Printed in Great Britain
EFFECTS OF CYSTEAMINE ADMINISTRATION ON PLASMA CONCENTRATION OF METABOLITES, PANCREATIC GLUCAGON AND INSULIN IN THE CHICKEN NICOLE RIDEAU*$, HUBERT KARMANN~ and JEAN SIMON* *Station de Recherches Avicoles, INRA, 37380 Nouzilly, France. Telephone: 4742-7700; TLaboratoire d’Etude des RCgulations Physiologiques, CNRS 23 Rue Becquerel, 67087 Strasbourg, France (Received 7 December
1989)
Abstract-l. The effects of subcutaneous injection of cysteamine (2-mercaptoethylamine, 300 mg/kg) were investigated in 54 week-old chickens. 2. In the short term (1 hr), cysteamine increased plasma levels of glucose, free fatty acids and insulin, and decreased that of a-amino non protein nitrogen. 3. In a longer term (17-24 hr), cysteamine increased the plasma level of glucose, did not modify those of a-amino non protein nitrogen, insulin and glucagon and decreased that of free fatty acids. 4. The disposal of an oral glucose load was impaired and the glucose-induced inhibition of pancreatic glucagon and stimulation of insulin release were blunted 17 hr after cysteamine administration. 5. Therefore, cysteamine exerts multiple effects on chicken pancreatic islet cells.
MATERIALS AND METHODS
INTRODUflION In the rat, cysteamine (2 mercaptoethylamine) which HBS initially used as an ulcerogenic agent, has been
shown to rapidly and reversibly deplete somatostatin (SRIF) stores from the plasma, and the cells of digestive tract, hypothalamus and pancreas following irr viva injection (Szabo and Reichlin, 1981). Cysteamine also exerts various effects on in viuo and in vitro glucagon and insulin release in rodents (Brown 6’1al., 1983; Sorenson et al., 1983; Ostenson and Efendic, 1985; Pate1 et al., 1985; Petersson and Hellerstriim, 1985; Takahashi et al., 1985; Sako et al., 1986; Silvestre et al., 1986; Petersson, 1987). It is still unclear whether or not those effects are only the consequence of the disappearance of pancreatic somatostatin. In chickens as in mammals, somatostatin is likely to exert a permanent inhibitory action on the pancreas. Such a possibility has been suggested by the fact that in isolated chicken pancreas, perfusion of anti-SRIF antiserum increases whereas perfusion 01’ exogenous SRIF decreases insulin and glucagon release (Honey et al., 1980, 1981). To our knowledge, the effects of cysteamine on plasma pancreatic glucagon and insulin have not yet been examined in chickens. Only one study (Zavy and Lindsey, 1988) reports a decrease of food intake without any alteration of body weight gain and an increase of gizzard ulceration incidence following incorporation of cysteamine in the diet of growing chickens. In the present study, the short term effects of subcutaneous injection of cysteamine on plasma nutrients and in vim pancreatic reponsiveness were examined in chickens. $To whom correspondence
should be addressed.
Animals and experimental conditions
Day-old male chickens (Shaver) were obtained from a commercial hatchery. They were brooded in conventional floor pens from day 1 to 4 weeks of age and then transferred in individual wire cages in a controlled environment (temperature and humidity) room and fed ad libitum a regular and balanced diet. The time course of the effects of subcutaneous injections (pectoral area) of cysteamine on plasma nutrient levels and pancreatic hormones was first examined. In a preliminary experiment (data not shown), the dose of 300 mg cysteamine (M 6500 Sigma) per kg of body weight induced a large hyperglycaemic effect without any mortality and was therefore chosen in the subsequent experiments. At 6 weeks of age, seven groups of 8 chickens homogenous for body weight were used. Three groups received saline as controls and three groups received cysteamine in the fed state. Following injection, chickens were fasted and blood samples (5-10 ml) were taken from the wing vein at either 30 min and 4 hr, 1 and 24 hr or only 2 hr so that each chicken was sampled twice, at most. At the time of injections (time 0) blood samples were obtained from another group of eight chickens in the fed state. In a further experiment, the effect of cysteamine on the glucose tolerance was examined at 5 weeks of age. Seventeen hours before the oral glucose tolerance test, chickens were subcutaneously injected with either saline or cysteamine (300 mg/kg), then fasted overnight. A load of 2 g glucose/kg b.w. (using a 50% glucose solution w/v) was administered in the crop by oral intubation. Blood samples were obtained using 8 different chickens at each of the various times indicated in the result section. Plasma determinations and statistical analysis
Blood samples were taken from a wing vein using heparin as anticoagulant. Blood samples were chilled, centrifuged and aliquot samples of plasma were stored at - 20°C. Plasma glucose was determined by the glucose oxidase technique using a glucose analyser (Model 2, Beckman 327
328
NICOLE BDEAU
instruments, Palo Alto, CA). Plasma free fatty acids were extracted by the Dole method (Dole and Meinertz, 1960) and measured according to a semi-automatic calorimetric method (Fruchart et al., 1974) by using palmitic acid as the standard. Plasma a-amino non protein nitrogen level was determined after extraction with 10% a-sulfosalicyclic acid, with the ninhydrin coloration method by using alanin (with O.ls/w heparin added) as the standard (Moore and Stein, 1954). Plasma chicken insulin was measured by radioimmunoassay (RIA) using a guinea pig antiporcine insulin serum (a gift of G. Rosselin, HBpital Saint Antoine, Paris) and chicken insulin as the standard and the tracer (Simon et al., 1974). lmmunoreactive glucagon was determined by the RIA method of Leclercq-Meyer et al. (1970) using pork glucagon (Lilly) as a standard and a specific C-terminal pancreatic glucagon antibody from Leclercq-Meyer which does not cross-react with gut glucagon (Karmann, 1984). Data were analyzed using Student’s t test (Snedecor and Cochran, 1956).
chickens but was significantly decreased at 24 hr as compared to initial levels (P < 0.05 vs initial level = 731 f 31 pgg/ml in the fed state, see legend to Table 1). From 30min to 4 hr, cysteamine significantly decreased plasma a-amino non protein nitrogen levels as compared to control levels (P < 0.05 or 0.01 vs saline). Plasma pancreatic glucagon levels were not different between cysteamine treated and control chickens even in the early times (30min and 1 hr, Table 1). When it has been measured (30 min-2 hr) plasma insulin levels were in contrast significantly increased in cysteamine-treated chickens (P < 0.05 vs saline at 30min and 1 hr). Eflect of cysteamine on glucose tolerance (Fig. 1)
During the oral glucose tolerance test (Fig. 1) performed in the fasting state at 17 hr following cysteamine or saline injection, initial plasma nutrient levels exhibited changes closed to those observed in the previous experiment at 24 hr following the injection (see Table 1) i.e. glucose was increased whereas free fatty acids and a-amino non protein nitrogen (aNP) were decreased after cysteamine as compared to controls (P < 0.01 for glucose and free fatty acids, P > 0.05 for aNP); plasma glucagon and insulin were unchanged. Following the glucose load, plasma glucose increased to the same level at 30 min in cysteaminetreated and control chickens but remained at significantly higher levels at 90min in cysteamine-treated chickens (P < 0.01 vs saline). In control chickens, plasma free fatty acids were significantly decreased from 20 to 60 min (P < 0.01 vs time 0) and returned to initial levels at 90 min; in cysteamine-treated chickens, plasma free fatty acids surprisingly increased to the level of controls at 10 min (P < 0.05 vs time 0), then exhibited changes parallel to those observed in controls up to 60 min, and remained however significantly lower at 90 min (P < 0.01 vs saline). Plasma a-amino non protein nitrogen was decreased below initial levels from 45 min up to 90 min in controls (P < 0.05 or 0.01 vs time 0); in cysteamine-treated chickens, the decrease appeared earlier at 15 min (P < 0.01 vs time 0). From 10 to 30 min, the plasma
RESULTS
Time course of the eflects of cysteamine on plasma nutrients, glucagon and insulin (Table 1)
In control (saline treated) chickens, plasma glucose levels slightly decreased during the time course (Table 1, chickens were fasted at time 0) and at 24 hr were significantly lower than initial level measured in the fed state (P < 0.01 vs initial level = 2.27 f 0.05 g/l, see legend to Table 1). In cysteamine-treated chickens, plasma glucose increased above initial level as early as 30 min following injection (P < 0.01 vs initial level) and was significantly higher than in controls at each time during the time course (P < 0.01 or 0.05 vs saline). In control chickens, plasma free fatty acid levels increased above the initial level during the time course (P < 0.01 at 30 min, 4 and 24 hr; P < 0.05 at I hr vs initial level = 125 + 4pg equivalent/ml in the fed state, see legend to Table 1). Following cysteamine treatment, plasma free fatty acids were significantly increased above control levels from 1 to 4 hr (P < 0.05 or 0.01 vs saline). In contrast, at 24 hr they were significantly lower in cysteamine treated than in control chickens (P < 0.01 vs saline). Plasma 8 -amino non protein nitrogen did not exhibit large changes up to 4 hr in control Table
I, Time course of the effects of subcutaneous
Time following
injection
Plasma glucose (g/l) saline cysteamine Plasma free fatty acids (~gjml) saline cysteamine Plasma *-amino sahne cysteamine Plasma pancreatic saline cysteamme
non protein
glucagon
Plasma msuhn (ng/ml) saline cysteamine
nitrogen
administration
et al.
of cysteamine
(300 mg/kg) on plasma
nutrients,
glucagon
and insulin
30 min
1 hr
2 hrs
4hrs
24 hrs
2.40 + 0.08 (8) 3.13tfO.l3(8)
2.22 f 0.03 (8) 2.83’ + 0.20 (8)
2.22 + 0.05 (8) 3.00. k 0.24 (8)
2.23 + 0.06 (8) 3.45’ + 0.39 (8)
I .89 + 0.02 (8) 2. I9t + 0.07 (8)
I76 +_ 8 (8) 177 f I6 (8)
142 F 6(8) 220’ + 18 (8)
136 f 6 (8) 236t i 22 (8)
170 5 lO(8) 2317 + 7 (8)
180 + 9 (8) 122t * II (8)
(fig/ml) 774 k 36 (8) 637. + 32 (8)
738 + 68 (8) 578’ + I7 (8)
768 ?r 27 (8) 584t i 31 (8)
655 + 27 (8) 539. + 42 (8)
644+21(g) 566 + 70 (8)
(pg/ml) I.2 i 0.3 (6) 0.8 k 0.1 (6)
I.1 + 0.2 (6) 0.7 F 0.1 (6)
0.8 f 0.1 (7) 0.8 k 0.1 (6)
1.08 f 0.08 (8) 2.12* f 0.40(7)
0.88 k 0.16(6) I .72 + 0.28 (7)
1.32*0.16(8) 2.08 + 0.40 (8)
Note: Cysteamine or saline was injected (time 0) to 6 week-old-fed injection, initial plasma concentrations were: glucose, 2.27 + 73 I + 3 I pgiml; pancreatic glucagon: 0.60 k 0.06 pg/ml; insulin parentheses (*.P < 0.05; t,P < 0.01 cysteamine vs saline using
I .4 i 0.2 (7) 1.0~0.1 (6) ND ND
I.1 50.1 (8) l.2&0.1 (6) ND ND
chickens which were then fasted during the time course. At the time of 0.05 g/l; free fatty acids, I25 f 4 pg/ml; a-amino no protein nitrogen, was not determined. Means + SEM with number of chickens given in Student’s I test; (ND) not determined).
Cysteamine and chicken metabolism
s ;;
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CYSTEAtllNE (300mg/kg)
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20
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60
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140
$
120
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82
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I
r-*-r’r-r~.‘l‘.-*-f
20
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90
TIME (min)
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90
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0
10
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60
70
80
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TIME (min)
Fig. 1. Effect of cysteamine (3OOmg/kg SC) on glucose tolerance. Cysteamine was administered 17 hr before the oral glucose load (2 g/kg b.w.) to 5 weeks old chickens. Birds were fed at the time of cysteamine injection and then fasted overnight. Means + SEM, n = 5-8 different chickens at each time (*,P < 0.05; **,P +=z 0.01 cysteamine vs saline) using Student’s I test).
n-amino non protein nitrogen level was lower in cysteamine-treated than in control chickens (P c 0.05 or 0.01); thereafter, it was similar in both groups. Plasma pancreatic glucagon levels were depressed following glucose from 10 to 45 min in controls (P < 0.05 or 0.01 vs time 0) and re-achieved initial levels at 90 min; in cysteamine-treated chickens, plasma pancreatic glucagon level was also depressed at 1Omin (P < 0.01 vs time 0), but recovered high levels, not significantly different from initial levels as early as 1.5min (P > 0.05 vs time 0). Plasma insulin levels increased above fasting levels in response to
glucose from IO to 45 min in control chickens (P c 0.01 vs time 0) and in contrast only weakly and not signi~cantly at 10 min in cysteamine-treated chickens (P > 0.05 vs time 0). DISCUSSION
As observed in the present experiments, cysteamine exerts multiple short term effects in young growing chickens; increases in plasma glucose, free fatty acids and insulin and decreases in plasma a-amino nitrogen. Most of these effects disappear rapidly; however
NICOLERIDEAU
330
an hyperglycaemic state persists and an hypo free fatty acid state develops at 17-24 hr. Those changes are close to those observed in mammals following cysteamine (Brown et al., 1983; Kabayama et al., 1985) and in chickens following in uivo passive immunoneutralization of somatostatin (Hall et al., 1986). Whether or not those effects are exerted through the disappearance of somatostatin itself or through the influence of other hormones following the disappearance of somatostatin is at present unknown. Active neoglucogenesis most likely from amino-acids and active lipolysis are suggested following cysteamine injection. Even though glucagon is the main, if not the unique, lipolytic hormone in birds (Langslow and Hales, 1969) those changes are not dependent on pancreatic glucagon since this hormone does not increase after cysteamine. Other hyperglycaemic hormones, possibly GH following the clearing of the inhibitory SRIF action and/or corticosterone as a stress reponse may be involved. Both hormones are effectively, acutely and transiently increased following injection of antiSRIF antibody in young cockerels (Harvey et ai., 1986; Cheung et al., 1988). The transient increase in plasma insulin levels, which was also observed in rats 4 hr after S.C.administration of cysteamine (Brown et al., 1983), may have several origins: a direct effect of cysteamine on /lcells, the clearing of the tonic somatostatin inhibitory effect which surprisingly would not apply to glucagon producing cells, a response to the hyperglycaemic state and/or the development of tissue insulin resistance. This situation is however transient since 17 hr after cysteamine, plasma insulin levels are not different from those of controls despite the presence of a hyperglycaemia. Following cysteamine, the glucose tolerance exhibits several alterations including an impairment of the glucose disposal, a delayed reappearance of free fatty acids despite high plasma glucagon levels (which are not suppressed by glucose) and the blunting of glucose-induced insulin release. The latter effect very likely accounts for the impairment of the glucose disposal. The origin of such alterations is difficult to investigate and identify. However, it can be concluded that in chickens cysteamine did not promote insulin release from the /I-cells as would have been expected if depletion of SRIF stores in pancreatic D-cells was the unique effect of cysteamine and that in uivo, cysteamine is likely to exert multiple effects in chicken pancreatic islet cells and other endocrine systems. Acknowledgements-The
authors are grateful to MarieFrance Scheller for her excellent technical work. The advices
given by Bernadette Chevalier and Gerard Guy for the measurements of plasma cc-amino non protein nitrogen and free fatty acids, respectively are acknowledged. Jean Marie Hervouet and Raymond Bonsergent for expert care of the animals, Christine Lessire for typing the manuscript are sincerely thanked. REFERENCES Brown M.R., Fisher L. A., Sawchenko P. E., Swanson L. W. and Vale W. W. (1983) Biological effects of cysteamine: relationship to somatostatin depletion. Reg. Pept. 5, 163-179.
et
al.
Cheung A., Harvey S., Hall T. R., Lam S. K. and Spencer G. S. G. (1988) Effects of passive immunization with antisomatostatin serum on plasma corticosterone concentrations in young domestic cockerels. J. Endocr. 116, 1799183.
Dole V. P. and Meinertz H. (1960) Microdetermination of long chain fatty acids in plasma and tissues. J. biol. Chem. 235, 2595-2599.
Fruchart J. C., Dewailly P., Jaillard J. and Sezille G. (1974) Dosage colorimetrique semi-automatique des acides gras. Ann. Biol. clin. 32, 237-244.
Hall T. R., and Spencer neutralization the domestic
Cheung A., Harvey S., Lam S. K. G. S. G. (1986) Somatostatin immunoaffects plasma metabolite concentrations in fowl. Comp. Biochem. Physiol. 85A (3)
489494.
Harvey S., Lam S. K. and Hall T. R. (1986) Somatostatin tonically inhibits growth hormone secretion in domestic fowl. J. Endocr. 111, 91-97. Honey R. N., Arimura A. and Weir G. C. (1981) Somatostatin neutralization stimulates glucagon and insulin secretion from the avian pancreas. Endocrinology 109, 1971-1974. Honey R. N., Fallon M. B. and Weir G. C. (1980) Effects of exogenous insulin, ghrcagon, and somatostatin on islet hormone secretion in the perfused chicken pancreas. Metabolism 29, 1242-1246. Kabayamay Y., Kato Y., Tojo K., Shimatsu A., Ohta H. and Imura H. (1985) Central effects of DN1417, a novel TRH analog, on plasma glucose and catecholamines in conscious rats. Life Sci. 36, 1287-1294.
Karmann H. (1984) Le diab&e experimental chez l’oie. Role de l’insuline dans la reponse du glucagon pancreatique et du gut GLI, et dans la regulation des acides amints plasmatiques. These Docteur es Sciences, Strasbourg. Langslow D. R. and Hales C. N. (1969) Lipolysis in chicken adipose tissue in vitro. J. Endocr. 43, 285-294. Leclercq-Meyer V., Mialhe P. and Malaisse W. J. (1970) Une methode de dosage radioimmunologique du glucagon comportant une separation par charbon dextran. Diabetologia 6, 121-129. Moore S. and Stein W. H. (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. biol. Chem. 211, 907-913. Ostenson C. G. and Efendic S. (1985) Cysteamine exerts multiple effects on the endocrine cells of the rat pancreas. Biochim. biophys. Acta. 846, 2422247.
Pate1 Y. C., Pierzchala I., Amberdt M. and Orci L. (1985) Effects of cysteamine and antibody to somatostatin on islet cell function in vitro. Evidence that intracellular somatostatin deficiency augments insulin and glucagon secretion. J. clin. Invest. 75, 124991255. Petersson C. (1987) The effect of cysteamine on the somatostatin content of guinea-pig islets. Up&a J. Med. Sci. 92, 85-88. Petersson B. and Hellerstrom C. (1985) Rapid depletion of somatostatin in isolated mouse pancreatic islets after treatment with cysteamine. Acta Endocrinol. 110, 227-23 1. Sako Y., Wasada T., Umeda F. and Ibayashi H. (1986) Effect of glibenclamide on pancreatic hormone release from isolated perifused islets of normal and cysteamine treated rats. Metabolism 35, 944-949. Silverstre R. A., Miralles P., Moreno P., Villanueva M. L. and Marco J. (1986) Somatostatin, insulin and glucagon secretion by the perfused pancreas from the cysteaminetreated rat. Biochem. biophys. Res. Commun. 134, 1291-1297. Simon J., Freychet P. and Rosselin G. (1974) Chicken characterization and insulin: radioimmunological
Cysteamine and chicken metabolism
331
enhanced activity in rat fat cells and liver plasma (1985) Effect of cysteamine on the secretion of pancreatic membranes. E~do~ri~~~ogy 95, 1439-1449. ho~on~ by the isolated perfused rat pancreas. Biomed. Snedecor G. W. and Cochran W. G. (1956) S~a~~~ica~ l&s. &, Suppl: 59-62. Methods, Ed 5. p. 100. The Iowa State College Press, Szabo S. and Reichlin S. (1981) Somatostatin in rat tissues Ames, I. A. is depleted by cysteamine administration. Endocrinology 109, 225-2257. Sorenson R. L., Grouse L. H. and Elder R. P. (1983) Cysteamine blocks somatostatin secretion without Zavy M. T. and Lindsey T. 0. (1988) Effect of cysteamine administration on growth and efficiency of altering the course of insulin or glucagon release. Diuberes food utilisation in chicken. 5rit. Pot&. Sci. 29, 32, 377-379. 4094 I 7. Takahasi K., Hara M., Sato N., Yamatani K. and Sasaki H.
0300-9629/90$3.00+ 0.00 iQ 1990Pergamon Press plc
Printed in Great Britain
EFFECTS OF CYSTEAMINE ADMINISTRATION ON PLASMA CONCENTRATION OF METABOLITES, PANCREATIC GLUCAGON AND INSULIN IN THE CHICKEN NICOLE RIDEAU*$, HUBERT KARMANN~ and JEAN SIMON* *Station de Recherches Avicoles, INRA, 37380 Nouzilly, France. Telephone: 4742-7700; TLaboratoire d’Etude des RCgulations Physiologiques, CNRS 23 Rue Becquerel, 67087 Strasbourg, France (Received 7 December
1989)
Abstract-l. The effects of subcutaneous injection of cysteamine (2-mercaptoethylamine, 300 mg/kg) were investigated in 54 week-old chickens. 2. In the short term (1 hr), cysteamine increased plasma levels of glucose, free fatty acids and insulin, and decreased that of a-amino non protein nitrogen. 3. In a longer term (17-24 hr), cysteamine increased the plasma level of glucose, did not modify those of a-amino non protein nitrogen, insulin and glucagon and decreased that of free fatty acids. 4. The disposal of an oral glucose load was impaired and the glucose-induced inhibition of pancreatic glucagon and stimulation of insulin release were blunted 17 hr after cysteamine administration. 5. Therefore, cysteamine exerts multiple effects on chicken pancreatic islet cells.
MATERIALS AND METHODS
INTRODUflION In the rat, cysteamine (2 mercaptoethylamine) which HBS initially used as an ulcerogenic agent, has been
shown to rapidly and reversibly deplete somatostatin (SRIF) stores from the plasma, and the cells of digestive tract, hypothalamus and pancreas following irr viva injection (Szabo and Reichlin, 1981). Cysteamine also exerts various effects on in viuo and in vitro glucagon and insulin release in rodents (Brown 6’1al., 1983; Sorenson et al., 1983; Ostenson and Efendic, 1985; Pate1 et al., 1985; Petersson and Hellerstriim, 1985; Takahashi et al., 1985; Sako et al., 1986; Silvestre et al., 1986; Petersson, 1987). It is still unclear whether or not those effects are only the consequence of the disappearance of pancreatic somatostatin. In chickens as in mammals, somatostatin is likely to exert a permanent inhibitory action on the pancreas. Such a possibility has been suggested by the fact that in isolated chicken pancreas, perfusion of anti-SRIF antiserum increases whereas perfusion 01’ exogenous SRIF decreases insulin and glucagon release (Honey et al., 1980, 1981). To our knowledge, the effects of cysteamine on plasma pancreatic glucagon and insulin have not yet been examined in chickens. Only one study (Zavy and Lindsey, 1988) reports a decrease of food intake without any alteration of body weight gain and an increase of gizzard ulceration incidence following incorporation of cysteamine in the diet of growing chickens. In the present study, the short term effects of subcutaneous injection of cysteamine on plasma nutrients and in vim pancreatic reponsiveness were examined in chickens. $To whom correspondence
should be addressed.
Animals and experimental conditions
Day-old male chickens (Shaver) were obtained from a commercial hatchery. They were brooded in conventional floor pens from day 1 to 4 weeks of age and then transferred in individual wire cages in a controlled environment (temperature and humidity) room and fed ad libitum a regular and balanced diet. The time course of the effects of subcutaneous injections (pectoral area) of cysteamine on plasma nutrient levels and pancreatic hormones was first examined. In a preliminary experiment (data not shown), the dose of 300 mg cysteamine (M 6500 Sigma) per kg of body weight induced a large hyperglycaemic effect without any mortality and was therefore chosen in the subsequent experiments. At 6 weeks of age, seven groups of 8 chickens homogenous for body weight were used. Three groups received saline as controls and three groups received cysteamine in the fed state. Following injection, chickens were fasted and blood samples (5-10 ml) were taken from the wing vein at either 30 min and 4 hr, 1 and 24 hr or only 2 hr so that each chicken was sampled twice, at most. At the time of injections (time 0) blood samples were obtained from another group of eight chickens in the fed state. In a further experiment, the effect of cysteamine on the glucose tolerance was examined at 5 weeks of age. Seventeen hours before the oral glucose tolerance test, chickens were subcutaneously injected with either saline or cysteamine (300 mg/kg), then fasted overnight. A load of 2 g glucose/kg b.w. (using a 50% glucose solution w/v) was administered in the crop by oral intubation. Blood samples were obtained using 8 different chickens at each of the various times indicated in the result section. Plasma determinations and statistical analysis
Blood samples were taken from a wing vein using heparin as anticoagulant. Blood samples were chilled, centrifuged and aliquot samples of plasma were stored at - 20°C. Plasma glucose was determined by the glucose oxidase technique using a glucose analyser (Model 2, Beckman 327
328
NICOLE BDEAU
instruments, Palo Alto, CA). Plasma free fatty acids were extracted by the Dole method (Dole and Meinertz, 1960) and measured according to a semi-automatic calorimetric method (Fruchart et al., 1974) by using palmitic acid as the standard. Plasma a-amino non protein nitrogen level was determined after extraction with 10% a-sulfosalicyclic acid, with the ninhydrin coloration method by using alanin (with O.ls/w heparin added) as the standard (Moore and Stein, 1954). Plasma chicken insulin was measured by radioimmunoassay (RIA) using a guinea pig antiporcine insulin serum (a gift of G. Rosselin, HBpital Saint Antoine, Paris) and chicken insulin as the standard and the tracer (Simon et al., 1974). lmmunoreactive glucagon was determined by the RIA method of Leclercq-Meyer et al. (1970) using pork glucagon (Lilly) as a standard and a specific C-terminal pancreatic glucagon antibody from Leclercq-Meyer which does not cross-react with gut glucagon (Karmann, 1984). Data were analyzed using Student’s t test (Snedecor and Cochran, 1956).
chickens but was significantly decreased at 24 hr as compared to initial levels (P < 0.05 vs initial level = 731 f 31 pgg/ml in the fed state, see legend to Table 1). From 30min to 4 hr, cysteamine significantly decreased plasma a-amino non protein nitrogen levels as compared to control levels (P < 0.05 or 0.01 vs saline). Plasma pancreatic glucagon levels were not different between cysteamine treated and control chickens even in the early times (30min and 1 hr, Table 1). When it has been measured (30 min-2 hr) plasma insulin levels were in contrast significantly increased in cysteamine-treated chickens (P < 0.05 vs saline at 30min and 1 hr). Eflect of cysteamine on glucose tolerance (Fig. 1)
During the oral glucose tolerance test (Fig. 1) performed in the fasting state at 17 hr following cysteamine or saline injection, initial plasma nutrient levels exhibited changes closed to those observed in the previous experiment at 24 hr following the injection (see Table 1) i.e. glucose was increased whereas free fatty acids and a-amino non protein nitrogen (aNP) were decreased after cysteamine as compared to controls (P < 0.01 for glucose and free fatty acids, P > 0.05 for aNP); plasma glucagon and insulin were unchanged. Following the glucose load, plasma glucose increased to the same level at 30 min in cysteaminetreated and control chickens but remained at significantly higher levels at 90min in cysteamine-treated chickens (P < 0.01 vs saline). In control chickens, plasma free fatty acids were significantly decreased from 20 to 60 min (P < 0.01 vs time 0) and returned to initial levels at 90 min; in cysteamine-treated chickens, plasma free fatty acids surprisingly increased to the level of controls at 10 min (P < 0.05 vs time 0), then exhibited changes parallel to those observed in controls up to 60 min, and remained however significantly lower at 90 min (P < 0.01 vs saline). Plasma a-amino non protein nitrogen was decreased below initial levels from 45 min up to 90 min in controls (P < 0.05 or 0.01 vs time 0); in cysteamine-treated chickens, the decrease appeared earlier at 15 min (P < 0.01 vs time 0). From 10 to 30 min, the plasma
RESULTS
Time course of the eflects of cysteamine on plasma nutrients, glucagon and insulin (Table 1)
In control (saline treated) chickens, plasma glucose levels slightly decreased during the time course (Table 1, chickens were fasted at time 0) and at 24 hr were significantly lower than initial level measured in the fed state (P < 0.01 vs initial level = 2.27 f 0.05 g/l, see legend to Table 1). In cysteamine-treated chickens, plasma glucose increased above initial level as early as 30 min following injection (P < 0.01 vs initial level) and was significantly higher than in controls at each time during the time course (P < 0.01 or 0.05 vs saline). In control chickens, plasma free fatty acid levels increased above the initial level during the time course (P < 0.01 at 30 min, 4 and 24 hr; P < 0.05 at I hr vs initial level = 125 + 4pg equivalent/ml in the fed state, see legend to Table 1). Following cysteamine treatment, plasma free fatty acids were significantly increased above control levels from 1 to 4 hr (P < 0.05 or 0.01 vs saline). In contrast, at 24 hr they were significantly lower in cysteamine treated than in control chickens (P < 0.01 vs saline). Plasma 8 -amino non protein nitrogen did not exhibit large changes up to 4 hr in control Table
I, Time course of the effects of subcutaneous
Time following
injection
Plasma glucose (g/l) saline cysteamine Plasma free fatty acids (~gjml) saline cysteamine Plasma *-amino sahne cysteamine Plasma pancreatic saline cysteamme
non protein
glucagon
Plasma msuhn (ng/ml) saline cysteamine
nitrogen
administration
et al.
of cysteamine
(300 mg/kg) on plasma
nutrients,
glucagon
and insulin
30 min
1 hr
2 hrs
4hrs
24 hrs
2.40 + 0.08 (8) 3.13tfO.l3(8)
2.22 f 0.03 (8) 2.83’ + 0.20 (8)
2.22 + 0.05 (8) 3.00. k 0.24 (8)
2.23 + 0.06 (8) 3.45’ + 0.39 (8)
I .89 + 0.02 (8) 2. I9t + 0.07 (8)
I76 +_ 8 (8) 177 f I6 (8)
142 F 6(8) 220’ + 18 (8)
136 f 6 (8) 236t i 22 (8)
170 5 lO(8) 2317 + 7 (8)
180 + 9 (8) 122t * II (8)
(fig/ml) 774 k 36 (8) 637. + 32 (8)
738 + 68 (8) 578’ + I7 (8)
768 ?r 27 (8) 584t i 31 (8)
655 + 27 (8) 539. + 42 (8)
644+21(g) 566 + 70 (8)
(pg/ml) I.2 i 0.3 (6) 0.8 k 0.1 (6)
I.1 + 0.2 (6) 0.7 F 0.1 (6)
0.8 f 0.1 (7) 0.8 k 0.1 (6)
1.08 f 0.08 (8) 2.12* f 0.40(7)
0.88 k 0.16(6) I .72 + 0.28 (7)
1.32*0.16(8) 2.08 + 0.40 (8)
Note: Cysteamine or saline was injected (time 0) to 6 week-old-fed injection, initial plasma concentrations were: glucose, 2.27 + 73 I + 3 I pgiml; pancreatic glucagon: 0.60 k 0.06 pg/ml; insulin parentheses (*.P < 0.05; t,P < 0.01 cysteamine vs saline using
I .4 i 0.2 (7) 1.0~0.1 (6) ND ND
I.1 50.1 (8) l.2&0.1 (6) ND ND
chickens which were then fasted during the time course. At the time of 0.05 g/l; free fatty acids, I25 f 4 pg/ml; a-amino no protein nitrogen, was not determined. Means + SEM with number of chickens given in Student’s I test; (ND) not determined).
Cysteamine and chicken metabolism
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Fig. 1. Effect of cysteamine (3OOmg/kg SC) on glucose tolerance. Cysteamine was administered 17 hr before the oral glucose load (2 g/kg b.w.) to 5 weeks old chickens. Birds were fed at the time of cysteamine injection and then fasted overnight. Means + SEM, n = 5-8 different chickens at each time (*,P < 0.05; **,P +=z 0.01 cysteamine vs saline) using Student’s I test).
n-amino non protein nitrogen level was lower in cysteamine-treated than in control chickens (P c 0.05 or 0.01); thereafter, it was similar in both groups. Plasma pancreatic glucagon levels were depressed following glucose from 10 to 45 min in controls (P < 0.05 or 0.01 vs time 0) and re-achieved initial levels at 90 min; in cysteamine-treated chickens, plasma pancreatic glucagon level was also depressed at 1Omin (P < 0.01 vs time 0), but recovered high levels, not significantly different from initial levels as early as 1.5min (P > 0.05 vs time 0). Plasma insulin levels increased above fasting levels in response to
glucose from IO to 45 min in control chickens (P c 0.01 vs time 0) and in contrast only weakly and not signi~cantly at 10 min in cysteamine-treated chickens (P > 0.05 vs time 0). DISCUSSION
As observed in the present experiments, cysteamine exerts multiple short term effects in young growing chickens; increases in plasma glucose, free fatty acids and insulin and decreases in plasma a-amino nitrogen. Most of these effects disappear rapidly; however
NICOLERIDEAU
330
an hyperglycaemic state persists and an hypo free fatty acid state develops at 17-24 hr. Those changes are close to those observed in mammals following cysteamine (Brown et al., 1983; Kabayama et al., 1985) and in chickens following in uivo passive immunoneutralization of somatostatin (Hall et al., 1986). Whether or not those effects are exerted through the disappearance of somatostatin itself or through the influence of other hormones following the disappearance of somatostatin is at present unknown. Active neoglucogenesis most likely from amino-acids and active lipolysis are suggested following cysteamine injection. Even though glucagon is the main, if not the unique, lipolytic hormone in birds (Langslow and Hales, 1969) those changes are not dependent on pancreatic glucagon since this hormone does not increase after cysteamine. Other hyperglycaemic hormones, possibly GH following the clearing of the inhibitory SRIF action and/or corticosterone as a stress reponse may be involved. Both hormones are effectively, acutely and transiently increased following injection of antiSRIF antibody in young cockerels (Harvey et ai., 1986; Cheung et al., 1988). The transient increase in plasma insulin levels, which was also observed in rats 4 hr after S.C.administration of cysteamine (Brown et al., 1983), may have several origins: a direct effect of cysteamine on /lcells, the clearing of the tonic somatostatin inhibitory effect which surprisingly would not apply to glucagon producing cells, a response to the hyperglycaemic state and/or the development of tissue insulin resistance. This situation is however transient since 17 hr after cysteamine, plasma insulin levels are not different from those of controls despite the presence of a hyperglycaemia. Following cysteamine, the glucose tolerance exhibits several alterations including an impairment of the glucose disposal, a delayed reappearance of free fatty acids despite high plasma glucagon levels (which are not suppressed by glucose) and the blunting of glucose-induced insulin release. The latter effect very likely accounts for the impairment of the glucose disposal. The origin of such alterations is difficult to investigate and identify. However, it can be concluded that in chickens cysteamine did not promote insulin release from the /I-cells as would have been expected if depletion of SRIF stores in pancreatic D-cells was the unique effect of cysteamine and that in uivo, cysteamine is likely to exert multiple effects in chicken pancreatic islet cells and other endocrine systems. Acknowledgements-The
authors are grateful to MarieFrance Scheller for her excellent technical work. The advices
given by Bernadette Chevalier and Gerard Guy for the measurements of plasma cc-amino non protein nitrogen and free fatty acids, respectively are acknowledged. Jean Marie Hervouet and Raymond Bonsergent for expert care of the animals, Christine Lessire for typing the manuscript are sincerely thanked. REFERENCES Brown M.R., Fisher L. A., Sawchenko P. E., Swanson L. W. and Vale W. W. (1983) Biological effects of cysteamine: relationship to somatostatin depletion. Reg. Pept. 5, 163-179.
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Cysteamine and chicken metabolism
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