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P.45 Calcitonin gene related peptide is an important secretagogue for rat intestinal L-cells
Results: CUNS (#mol/(min*100g)) increased stepwise from GH values: 4.51 _+0.89, Con: 7.53 _+1.35, StGH: 8.40 + 0.11 to St group: 11.69 _+0.83 (mean _+SD) (St significantly higher than all other groups and StGH significantly lower than St animals), mRNA levels for urea cycle enzymes in the liver increased after steroid treatment, whereas concomitant administration of GH decreased gene expression, even more so when administered alone. N-balance and N-contents of organs decreased after steroid treatment. Combined treatment tended to normalize this, whereas GH administered alone increased these values by 30% (P < 0.05). Conclusions: Steroid treatment increases conversion of amino-N into urea-N, decreases N-balance and organ N-contents and increases mRNA levels of urea cycle enzymes. Concomitant administration of GH normalizes these parameters indicating that the reduction of the steroid induced catabolism, seen after GH treatment, is partly due to changes in liver handling of circulating amino acids. This may be due to a direct effect on the expression of urea cycle enzymes as indicated by the changes in mRNA abundances.
stimulants. In order to investigate the influence of calcitonin gene-related peptide (CGRP), a neuropeptide that is located in the enteric nervous system, we used an isolated vascularly perfused rat ileum preparation. The isolated loop was perfused with a Krebs-Henseleit-buffer containing 25% washed bovine erythrocytes. GLP-1 was determined in the portal effluent by radioimmunoassay using a highly specific polyclonal antibody. Results: The intra-arterial infusion of CGRP 1 0 - 7 M induced significant GLP-1 secretion with an initial peak of 73.2 _+ 17.8 fmol/2 min and a following sustained release at a level of 42.8 _+ 15.7fmol/2min (basal: 14.9 + 2fmol/2min). The comparison of the additionally secreted GLP-1 with CGRP stimulation showed a strong dose-dependency ( 1 0 - 7 M : 402.8 _+ 162fmol/30min; 10-8M: 159 _+ 78.5 fmol/30 min: 10-9 M: 78.5 _+41.1 fmol/30 min). The additional infusion of atropine 1 0 - 5 M had no effect (371.1 + 84.1fmol/30min) whereas additional infusion of galanin 1 0 - 7 M dramatically reduced the secretion induced by CGRP 1 0 - 7 M (50.5 _+ 54.8fmol/30min). Interestingly, the additional luminal perfusion of glucose 5% was able to potentiate the CGRP-7M-induced GLP-1 release (1246.5 _+ 392 fmol/30 min). Conclusions: CGRP is an important secretagogue for intestinal L-cells, especially in the presence of an additional luminal stimulus with glucose. CGRP might be important in the complex GLP-1 secretion regulation.
P.44 Growth hormone and insulin-like growth factor I e n h a n c e in vitro bactericidal activity of human polymorphonuclear neutrophils T. Inoue, H. Saito*, Y. Hashiguchi, K. Fukatsu, M. T. Lin, T. Inaba, I. Han, S. Furukawa, T. Matsuda and T. Muto Department of Surgery 1 and *Surgical Center, University of Tokyo, Tokyo, Japan.
P.46 Muscle glutamine metabolism in dexamethasonetreated rats: effect of aging C. M o i n a r d 1, R. Minet 1'2, S. Walrandt, F. Villi6t, M. Ma~collett, P. Davott, J. Chopineau 2 and L. Cynober1 1 Biochemistry, 2 Clinical Pharmacy, Pharmacy School, CRNH, Clermont-Ferrand, France.
We previously reported that 6-day pretreatment with growth hormone (GH) or insulin-like growth factor I (IGF-I) improved host defence and survival in septic mice. The purpose of this study was to investigate the effects of GH and IGF-I on in vitro bactericidal activity of human polymerphonuclear neutrophils. Materials and methods: Venous blood was drawn from healthy volunteers. Polymorphonuclear neutrophils (PMNs) were isolated by the FicolI-Hypaque density gradient method. PMNs (2.5 x 10 s) were preincubated with medium (control), GH (100,1000ng/mL) or IGF-I (100, 1000 ng/mL) at 37°C for 3 h. Escherichia coil (1 x 106) and human blood type AB serum (0.1 mL) were added to the medium only (PMN (-)) or pretreated PMN cultures). These mixtures (total volume: 1 mL) were incubated at 37°C. A 100#1 sample of the mixture was removed at 30 and 120 min incubation and serially diluted in sterile distilled water to lyse PMNs. Dilutions were plated on to sheep blood agar plates and bacterial colony-forming units (cfu) were determined after an overnight incubation. E. coil-killing activity of PMNs was evaluated according to the following formulae: Percentage E. coil killed = (1 - the number of cfu for pretreated groups/the number of cfu for PMN(-))xl00 (%). Viable PMN counts in control, GH1000 and IGF-I 1000 groups were also determined at 120min incubation. Statistical analyses were done by oneway repeated measures ANOVA followed by contrast test. Results: Percentage E. coil killed (%) is shown below.
Incubation time Control
A lack of adaptation to aggression is a characteristic of aging. Since glutamine (GLN) plays a central role in the regulation of muscular protein turnover and since cortisol is a major regulator of GLN metabolism, we have hypothesized that the alteration in the response to glucoccrticoids could be responsible for a perturbation of GLN metabolism. This in turn could lead to a poor adaptation to stress in aging. Male Sprague-Dawley rats aged 24 months old (n = 20) or 3 months old (n = 20) were divided into 8 groups of 5 animals. Groups Go.75, Gt.5o, G2.50 received intraperitoneal injections 0.75, 1.50 or 2.50mg/kg of dexamethasone (DEX) respectively during 5 days. The G2.5oPF group received isovolumic NaCI and was pair-fed with G2.5o. Muscles rich in type I fibres (soleus), type II fibres (EDL) or mixed (gastrocnemius) were analysed for GLN, glutamine-synthetase (GS) and protein contents. Results for GLN contents (#mol/g; mean +_SD): Soleus Gastrocnemius EDL 3 months 24 months 3 months 24 months 3 months 24 n'tonths G2.50PF 9,7-+1,4 8.9-+1.1 4,3-+0.6 2.1-+0.3 6.9-+0.7 5.5-+0.8 G2.5o 8,0 -+ 0.4 ~ 9.0 -+ 0.6 2.5 -+ 0.2 ! 1.8 -+ 0.7 3.9 -+ 0.6 ~' 2.9 _4:0,7t* ANOVA + Newman Ke~ls test: t p < 0,05; t p < 0.01 versus G2.5OPF. Student's t-test: *P< 0.05 versus 3 months old.
IGF-11000
GH 100
GH 1000
Similar results were obtained using other doses of DEX. Whatever the dose of DEX injected and the type of muscle studied, GS activity (nmol/mn/g) increased in adult and old rats (old rats EDL: 906 _4:117 in G2.5o vs 381 _+32 in G2.soPF; adult rats EDL: 1025 _+145 in G2.5o vs 348 _+ 106 in G2.5OPF; P < 0.05). In plasma, GLN concentrations decreased only in old rats (G2.so: 782 _+166 v s G 2 . 5 0 e F : 1113 _+173 #mol/I; P < 0.0!5). Conclusions: Red and white muscles display different behaviour with regard to the response to DEX and according to the age of the animal. Lower plasma GLN concentrations in old, stressed rats could result from a decrease in muscle efflux leading to an insufficient provision of GLN to enterocytes and other GLN consumer tissues.
IGF-1100 (ng/mL)
30min 24.7_+4.6 25.3_+5.3 40.2-+3.3 26.5_+8.5 120 min 28.0-+10.0 41.6-+2.0t 52.6_+2.3* 49.5+_3.5* Values are means -+SEMs. *P< 0.05 vs control, tp < &08 vs control.
34.2_+3.8 47.7_+6.3*
The number of viable PMNs in the GH 1000 group was greater than that in the control group (control: 6.0 _+ 1.9, GH 1000:8.8 _+2.1 *, IGF-I 1000: 6.6 _+1.6 x 105/mL, *P = 0.05 vs control). The number of viable PMNs correlated with the percentage E. cofikilled (r= 0.70, P < 0.05). Conclusions: Both GH and IGF-I directly enhanced in vitro bactericidal activity of human PMNs. GH also preserved viability of PMNs during bacterial killing.
P.47 Parenteral nutrition increases IGF-I after abdominal surgery I. Tj~der, A. Th6rne, M. Tally, P. Ess6n and J. Wernerman Dept of Anaesthesiology and Intensive Care and Dept of Surgery at Huddinge University Hospital, and Dept of Molecular Medicine the Endocrine and Diabetes Unit, Karolinska Hospital, Stockholm, Sweden.
P.45 Calcitonin gene related peptide is an important secretagogue for rat intestinal L-cells C. H e r r m a n n - R i n k e , R. Arnold and B. GSke Dept of Internal Medicine, Philipps, University of Marburg, Marburg, Germany, The incretin hormone GLP-1, located in intestinal L-cells in the lower gut, is known to be released into the circulation shortly after meals and thereby induces insulin secretion. Since blood levels rise immediately, even before nutrients have reached the distal gut, a complex nervous regulation can be suspected. Besides vagal reflexes, bpmbesin-like peptides and gastric inhibitory peptide (GIP) have been considered as potent
Surgery initiates a catabolic state with negative nitrogen balance in the postoperative period, which is not altered by provision of total parenteral nutrition (TPN). Insulin-like growth factor-I (IGF-I) is a mediator of the anabolic action of growth hormone and can be used as an indicator of nutritional status, showing low levels after major surgery and in the criti35
stimulants. In order to investigate the influence of calcitonin gene-related peptide (CGRP), a neuropeptide that is located in the enteric nervous system, we used an isolated vascularly perfused rat ileum preparation. The isolated loop was perfused with a Krebs-Henseleit-buffer containing 25% washed bovine erythrocytes. GLP-1 was determined in the portal effluent by radioimmunoassay using a highly specific polyclonal antibody. Results: The intra-arterial infusion of CGRP 1 0 - 7 M induced significant GLP-1 secretion with an initial peak of 73.2 _+ 17.8 fmol/2 min and a following sustained release at a level of 42.8 _+ 15.7fmol/2min (basal: 14.9 + 2fmol/2min). The comparison of the additionally secreted GLP-1 with CGRP stimulation showed a strong dose-dependency ( 1 0 - 7 M : 402.8 _+ 162fmol/30min; 10-8M: 159 _+ 78.5 fmol/30 min: 10-9 M: 78.5 _+41.1 fmol/30 min). The additional infusion of atropine 1 0 - 5 M had no effect (371.1 + 84.1fmol/30min) whereas additional infusion of galanin 1 0 - 7 M dramatically reduced the secretion induced by CGRP 1 0 - 7 M (50.5 _+ 54.8fmol/30min). Interestingly, the additional luminal perfusion of glucose 5% was able to potentiate the CGRP-7M-induced GLP-1 release (1246.5 _+ 392 fmol/30 min). Conclusions: CGRP is an important secretagogue for intestinal L-cells, especially in the presence of an additional luminal stimulus with glucose. CGRP might be important in the complex GLP-1 secretion regulation.
P.44 Growth hormone and insulin-like growth factor I e n h a n c e in vitro bactericidal activity of human polymorphonuclear neutrophils T. Inoue, H. Saito*, Y. Hashiguchi, K. Fukatsu, M. T. Lin, T. Inaba, I. Han, S. Furukawa, T. Matsuda and T. Muto Department of Surgery 1 and *Surgical Center, University of Tokyo, Tokyo, Japan.
P.46 Muscle glutamine metabolism in dexamethasonetreated rats: effect of aging C. M o i n a r d 1, R. Minet 1'2, S. Walrandt, F. Villi6t, M. Ma~collett, P. Davott, J. Chopineau 2 and L. Cynober1 1 Biochemistry, 2 Clinical Pharmacy, Pharmacy School, CRNH, Clermont-Ferrand, France.
We previously reported that 6-day pretreatment with growth hormone (GH) or insulin-like growth factor I (IGF-I) improved host defence and survival in septic mice. The purpose of this study was to investigate the effects of GH and IGF-I on in vitro bactericidal activity of human polymerphonuclear neutrophils. Materials and methods: Venous blood was drawn from healthy volunteers. Polymorphonuclear neutrophils (PMNs) were isolated by the FicolI-Hypaque density gradient method. PMNs (2.5 x 10 s) were preincubated with medium (control), GH (100,1000ng/mL) or IGF-I (100, 1000 ng/mL) at 37°C for 3 h. Escherichia coil (1 x 106) and human blood type AB serum (0.1 mL) were added to the medium only (PMN (-)) or pretreated PMN cultures). These mixtures (total volume: 1 mL) were incubated at 37°C. A 100#1 sample of the mixture was removed at 30 and 120 min incubation and serially diluted in sterile distilled water to lyse PMNs. Dilutions were plated on to sheep blood agar plates and bacterial colony-forming units (cfu) were determined after an overnight incubation. E. coil-killing activity of PMNs was evaluated according to the following formulae: Percentage E. coil killed = (1 - the number of cfu for pretreated groups/the number of cfu for PMN(-))xl00 (%). Viable PMN counts in control, GH1000 and IGF-I 1000 groups were also determined at 120min incubation. Statistical analyses were done by oneway repeated measures ANOVA followed by contrast test. Results: Percentage E. coil killed (%) is shown below.
Incubation time Control
A lack of adaptation to aggression is a characteristic of aging. Since glutamine (GLN) plays a central role in the regulation of muscular protein turnover and since cortisol is a major regulator of GLN metabolism, we have hypothesized that the alteration in the response to glucoccrticoids could be responsible for a perturbation of GLN metabolism. This in turn could lead to a poor adaptation to stress in aging. Male Sprague-Dawley rats aged 24 months old (n = 20) or 3 months old (n = 20) were divided into 8 groups of 5 animals. Groups Go.75, Gt.5o, G2.50 received intraperitoneal injections 0.75, 1.50 or 2.50mg/kg of dexamethasone (DEX) respectively during 5 days. The G2.5oPF group received isovolumic NaCI and was pair-fed with G2.5o. Muscles rich in type I fibres (soleus), type II fibres (EDL) or mixed (gastrocnemius) were analysed for GLN, glutamine-synthetase (GS) and protein contents. Results for GLN contents (#mol/g; mean +_SD): Soleus Gastrocnemius EDL 3 months 24 months 3 months 24 months 3 months 24 n'tonths G2.50PF 9,7-+1,4 8.9-+1.1 4,3-+0.6 2.1-+0.3 6.9-+0.7 5.5-+0.8 G2.5o 8,0 -+ 0.4 ~ 9.0 -+ 0.6 2.5 -+ 0.2 ! 1.8 -+ 0.7 3.9 -+ 0.6 ~' 2.9 _4:0,7t* ANOVA + Newman Ke~ls test: t p < 0,05; t p < 0.01 versus G2.5OPF. Student's t-test: *P< 0.05 versus 3 months old.
IGF-11000
GH 100
GH 1000
Similar results were obtained using other doses of DEX. Whatever the dose of DEX injected and the type of muscle studied, GS activity (nmol/mn/g) increased in adult and old rats (old rats EDL: 906 _4:117 in G2.5o vs 381 _+32 in G2.soPF; adult rats EDL: 1025 _+145 in G2.5o vs 348 _+ 106 in G2.5OPF; P < 0.05). In plasma, GLN concentrations decreased only in old rats (G2.so: 782 _+166 v s G 2 . 5 0 e F : 1113 _+173 #mol/I; P < 0.0!5). Conclusions: Red and white muscles display different behaviour with regard to the response to DEX and according to the age of the animal. Lower plasma GLN concentrations in old, stressed rats could result from a decrease in muscle efflux leading to an insufficient provision of GLN to enterocytes and other GLN consumer tissues.
IGF-1100 (ng/mL)
30min 24.7_+4.6 25.3_+5.3 40.2-+3.3 26.5_+8.5 120 min 28.0-+10.0 41.6-+2.0t 52.6_+2.3* 49.5+_3.5* Values are means -+SEMs. *P< 0.05 vs control, tp < &08 vs control.
34.2_+3.8 47.7_+6.3*
The number of viable PMNs in the GH 1000 group was greater than that in the control group (control: 6.0 _+ 1.9, GH 1000:8.8 _+2.1 *, IGF-I 1000: 6.6 _+1.6 x 105/mL, *P = 0.05 vs control). The number of viable PMNs correlated with the percentage E. cofikilled (r= 0.70, P < 0.05). Conclusions: Both GH and IGF-I directly enhanced in vitro bactericidal activity of human PMNs. GH also preserved viability of PMNs during bacterial killing.
P.47 Parenteral nutrition increases IGF-I after abdominal surgery I. Tj~der, A. Th6rne, M. Tally, P. Ess6n and J. Wernerman Dept of Anaesthesiology and Intensive Care and Dept of Surgery at Huddinge University Hospital, and Dept of Molecular Medicine the Endocrine and Diabetes Unit, Karolinska Hospital, Stockholm, Sweden.
P.45 Calcitonin gene related peptide is an important secretagogue for rat intestinal L-cells C. H e r r m a n n - R i n k e , R. Arnold and B. GSke Dept of Internal Medicine, Philipps, University of Marburg, Marburg, Germany, The incretin hormone GLP-1, located in intestinal L-cells in the lower gut, is known to be released into the circulation shortly after meals and thereby induces insulin secretion. Since blood levels rise immediately, even before nutrients have reached the distal gut, a complex nervous regulation can be suspected. Besides vagal reflexes, bpmbesin-like peptides and gastric inhibitory peptide (GIP) have been considered as potent
Surgery initiates a catabolic state with negative nitrogen balance in the postoperative period, which is not altered by provision of total parenteral nutrition (TPN). Insulin-like growth factor-I (IGF-I) is a mediator of the anabolic action of growth hormone and can be used as an indicator of nutritional status, showing low levels after major surgery and in the criti35