Growth hormone and growth hormone secretagogue effects on nitrogen balance and urea synthesis in steroid treated rats

Design Five groups of rats were included: (1) free-fed controls (2) pair-fed controls (3) prednisolone (delcortol, 4 mg × kg −1 × day −1 ) (4) prednisolone and GH (1 mg × kg −1 × day −1 ) (5) prednisolone and Ipamorelin (0.5 mg × kg −1 × day −1 ). After seven days the hepatic capacity of urea-N synthesis (CUNS) was determined in parallel with measurements of liver mRNA levels of urea cycle enzymes, whole-body N-balance, and N-contents of various organs. Results Compared to pair-fed controls, prednisolone increased CUNS ( p < 0.01) as well as the expression of urea cycle genes ( p < 0.01), and decreased N-balance ( p < 0.01) as well as organ N-contents ( p < 0.05). Compared to prednisolone treated animals, co-administration of GH reduced CUNS by 33% ( p < 0.01), normalized urea cycle gene expression, improved N-balance 2.5-fold, and normalized or improved organ N-contents. In prednisolone treated rats Ipamorelin reduced CUNS by 20% ( p < 0.05), decreased the expression of urea cycle enzymes, neutralised N-balance, and normalized or improved organ N-contents. Conclusion Accelerated nitrogen wasting in the liver and other organs caused by prednisolone treatment was counteracted by treatment with either GH or its secretagogue Ipamorelin, though at the doses given less efficiently by the latter. This functional study of animals confirms that the GH secretagogue exerts GH related metabolic effects and may be useful in the treatment of steroid-induced catabolism. Abbreviation BW body weight CUNS capacity of urea-N synthesis GH growth hormone IGF-1 insulin-like growth factor I CPS carbamoyl phosphate synthetase OTC ornithine transcarboxylase ASS argininosuccinate synthetase ASL argininosuccinate lyase ARG arginase Keywords Growth hormone Growth hormone secretagogue Glucocorticoids Protein metabolism Amino acids Gene expression 1 Introduction Glucocorticoid treatment, when used in various disease states, is a frequent cause of catabolism in humans. Catabolism is caused both by an increase in body protein breakdown and by an accelerated hepatic degradation of amino acid nitrogen into urea nitrogen [1–3] . Growth hormone may correct protein wasting during glucocorticoid treatment by increasing net tissue uptake of amino acid nitrogen as well as by reducing hepatic degradation of amino acid nitrogen into urea nitrogen [2,4–7] . Ipamorelin is a synthetic growth hormone (GH) secretagogue with specific GH releasing properties [8,9] . In GH deficient and catabolic states, the use of secretagogues may be more clinically feasible than treatment with GH itself, since it is possible to administer some of these secretagogues orally [10,11] . However, it remains to be documented that the secretagogues actually exert nitrogen preserving effects during catabolism similar to that of growth hormone. We wanted to examine whether Ipamorelin exerts the same biological effects in rats as does GH, by counteracting both the increase in ability of urea synthesis and by reducing the parallel loss of organ nitrogen induced by glucocorticoid administration. 2 Materials and methods 2.1 Animals Female Wistar rats (total N = 80, body weight 200–210 g) (Møllegaard Breeding Centre, Eiby, Denmark) were housed at 22 ± 2 °C, 55 ± 10% relative humidity. Air was changed 8–10 times per hour at a 12-h light, 12-h dark cycle (06.30–18.30 light). The animals had free or controlled access to standard chow (Altromin diet # 1324, Chr. Petersen Ltd., Slagelse, Denmark) and free access to tap water. Each cage contained two rats, each rat was weighed daily during the study period. Food consumption was recorded every day. Two days before investigational procedures all rats were housed singly in metabolic cages in order to determine individual N-balance. 2.2 Protocols There were five study groups: two control groups and three intervention groups. All animals in the intervention groups were treated with prednisolone, GH, or Ipamorelin twice daily for seven days, and were given the last injection in the morning on the day of CUNS determinations. 1. Free-fed controls (Ff Con): animals had free access to fodder and were injected with saline ( N = 16). 2. Pair-fed controls (Pf Con): animals were pair-fed to the prednisolone treated animals and injected with saline daily ( N = 16). 3. Prednisolone treated animals (St): animals had free access to fodder and were injected with 4 mg × kg −1 × day −1 of prednisolone (Delcortol, LEO, Copenhagen, Denmark) ( N = 16). 4. Prednisolone and GH treated animals (St+GH): Animals were pair-fed to the prednisolone treated rats and were injected with 4 mg × kg −1 × day −1 of prednisolone and 1 mg × kg −1 × day −1 of GH (Norditropin, Novo Nordisk, Bagsværd, Denmark) ( N = 16). 5. Prednisolone and Ipamorelin treated animals (St+IPA): Animals were pair-fed to the prednisolone treated rats and were injected with 4 mg × kg −1 × day −1 of prednisolone and 0.5 mg × kg −1 × day −1 of Ipamorelin (NNC 26-0161, Novo Nordisk, Bagsværd, Denmark) ( N = 16). Thus, all groups treated with prednisolone ate the same amount of food. GH, Ipamorelin, and saline were given subcutaneously in order to standardize the applied stress of the injection. In the free-fed control group only food consumption and weight gain were recorded. Pilot and previous studies have established prednisolone doses and relevant duration of treatment that would result in a significant weight loss [12,13] . The GH doses used were equivalent to doses in hypophysectomised rats and similar to our previous study [12] . Ipamorelin was given in a dose with maximum GH and IGF-I stimulatory ability [9] . In eight animals of each study group the initial blood α-amino-N concentration (AAN), the capacity of urea-N synthesis (CUNS), and whole-body N-balance were determined. Furthermore, in eight other animals of each study group nitrogen contents (N-contents) of liver, kidney, the soleus muscle, and the long extensor digitorum muscle (EDL) were determined together with liver mRNA levels of the flux- and rate-limiting urea cycle enzymes carbamoyl phosphate synthetase (CPS) and argininosuccinate synthetase (ASS). 2.3 Experimental procedures 2.3.1 CUNS Following anaesthesia with a subcutaneous injection of fentanyl/fluanisone (Hypnorm®, Jansen Pharma, Birkeroed, Denmark) 0.75 ml × kg −1 and midazolam (Dormicum®, La Roche AG, Basel, Schwitzerland) 4 mg × kg −1 , a catheter (Neoflon 0.6 mm, Viggo-Spectramed, Helsingborg, Sweden) was inserted into the femoral vein for continuous infusion. A retroperitoneal bilateral nephrectomy was performed in all animals immediately before the investigation started, in order to facilitate determination of urea synthesis, see below [14] . This procedure in itself did not acutely influence the rate of urea synthesis [14] . Blood samples were taken from the retrobulbary venous plexus using heparinised micropipettes (Vitrex Laboratory Equipment, Horsens, Denmark). For determination of the substrate saturated capacity of urea-N synthesis (CUNS), alanine was administered according to body weight as a bolus of 0.8–1.1 ml of a 1120 mmol × l −1 solution in sterile water followed by constant infusion of 2.6–3.2 ml × h −1 of a 224 mmol × l −1 solution for 70 min by means of an injectomat (Perfusor Secura, Braun, Melsungen, Germany). Steady-state blood α-amino-N concentration was defined as fluctuations below 10% during at least 50 min of the study, and the alanine infusion was aimed at obtaining a steady-state total blood α-amino-N concentration between 7.3 mmol × l −1 and 11.6 mmol × l −1 . Blood was sampled (100 μl) at 10 min intervals after an initial equilibration period of 20 min for determination of blood urea and total α-amino-N. A total of 1 ml of blood was removed. This volume was compensated by the infusion of alanine. Organs were isolated immediately after decapitation and weighed after blotting on filter paper, then frozen in toto in liquid nitrogen and stored at −80 °C until analyses. In the soleus muscle predominantly type I muscle fibres are found, and in the long extensor digitorum muscle type II fibres predominate [15] . Non-muscular nitrogen was examined by contents in liver and kidneys. On the last two days of the investigation period the animals were housed in metabolic cages and the amount of food ingested by each animal was determined. Samples of quantitatively collected urine were analyzed for total nitrogen and urea contents and samples of faeces were analyzed for nitrogen contents. From these measurements the nitrogen balance (mmol × 24 h −1 ) was calculated. 2.3.2 mRNA determinations The expression of hepatic urea cycle enzyme genes can be assessed by quantifying their mRNA levels. This allows identification of regulation at the level of gene expression. About 200 mg of liver tissue from the left lobe was immediately placed in liquid nitrogen. Total RNA was isolated with a Promega kit Z 5110 (Madison, WI) based on the thiocyanate method according to the specification of the manufacturer. Specificity of all probes was ascertained by autoradiography of northern blots, showing hybridization signals at the expected sites. Slot blots were used for quantification of mRNA levels using a Schleicher & Schuell Minifold (Schleicher & Schuell Minifold, Dassel, Germany). After blotting, the filters were UV cross-linked in a stratalinker (Stratagene, La Jolla, CA). The CV%, estimated by loading the same extract to 6–10 wells, was 7% on average. During testing we changed the procedure and used hybridization of ribosomal RNA as an index of mRNA signal. This change in procedure did not affect the results. 2.3.3 Hybridization About 25 ng cDNA was labelled by random priming using the multiprime kit RPN 1601Z (Amersham, Braunschweig, Germany,) and eluted on NICK TM spin columns (Pharmacia, Uppsala, Sweden). Prehybridization was performed at 42 °C for 1 h in a solution of formamide 50% (Merck, Darmstadt, Germany); Denhard solution 10 × (Sigma, St. Louis, MO); Tris 0.05 M ph 7:4; NaCl 1 M; SDS 1%-w/v; Napyrophosphate 0.1%-w/v; and salmon sperm DNA 0.25 mg/ml (Sigma) which was sonicated and immersed in boiling water for 15 min and then added to the solution. Hybridization was performed at 42 °C for 16 h with the same solution (except the 0.25 mg/ml salmon sperm DNA) with the labelled probe added. Finally, filters were washed twice with SSC 0.1 × and SDS 0.5%-w/v at 65 °C for 30 min. Phosphoimaging was made on an Imaging Plate BASIII TM (Fuji Photo Film, Kanagawa, Japan) under lead shield and the hybridization signal analyzed in a Fujix Bioimaging Analyzer System BAS2000 TM (Fuji Photo Film). 2.3.4 cDNA probes Carbamoyl phosphate synthetase I, pCPSr Pst 850 b [16] ; ornithine transcarbamylase pOTC1 HindIII 388 b [17] ; argininosuccinate synthetase pASr11 PstI 1450 b [18] ; argininosuccinate lyase AL-2 EcoR1 1000 b [19] ; and arginase 3B1 Pst1 650 b [20] . DNA fragments were separated by agarose gel electrophoresis and eluted on Spin bind DNA Extraction Units (FMC, Rockland, ME). Results were recorded based on optical density converted into percent, relative to pair-fed control values. 2.4 Analyses Blood urea concentration was measured by the urease-Berthelot method [21] and total blood α-amino-N concentration by the dinitroflourobenzene method [22] . Serum IGF-I concentration was measured by an in-house RIA using a polyclonal rabbit antibody (Nichols Institute Diagnostics, San Capistiano, Ca) and recombinant human IGF-I as standard (Amersham International, Amersham, Bucks, UK). All samples were analyzed in triplicates in one assay. Organ N-contents were determined by the micro Kjeldahl technique as previously described [23] . 2.5 Calculations Nitrogen balance equaled (food amount (g) × food nitrogen concentration (mmol × g −1 )) – (urine total nitrogen + faeces total nitrogen contents). The substrate-standardized capacity of urea-N synthesis (CUNS) (μmol × min −1 × 100 g BW −1 ) was calculated as the body accumulation of urea corrected for intestinal hydrolysis [14] . CUNS equaled dcu/dt × 0.63BW × 1/(1–0.11), where dcu/dt was the slope of the linear regression analysis of blood urea concentration at the time of steady-state. 0.63 BW (body weight) was the distribution volume of urea [24] and 1/(1–0.11) was the reported correction factor for intestinal hydrolysis and recycling of nitrogen from newly synthesized urea [14] . 2.6 Statistical methods Results are given as mean ± standard error of the mean (SE). The results were analyzed using one way analysis of variance (ANOVA) followed by Student–Newman–Keuls method for multiple comparisons, when appropriate, or a one sample t -test. A two-tailed p value < 0.05 was considered significant. 3 Results 3.1 Food intake Free-fed control rats ate 16 g daily on average. Prednisolone treated animals decreased their intake to 13 g daily. GH and Ipamorelin treated animals were pair-fed to prednisolone treated animals and thus received 13 g food daily. 3.2 Body weight Initially, all groups had identical body weights, data not shown. 3.3 Basal blood amino-N concentration ( Fig. 1 ) Prednisolone decreased basal blood α-amino-N concentration by 30% compared to pair-fed controls (3.0 ± 0.5 mmol × l −1 vs. 4.2 ± 0.5 mmol × l −1 , p < 0.05). Compared to steroid treated animals, the basal blood α-amino-N concentrations were higher in GH and Ipamorelin treated animals (3.0 ± 0.5 mmol × l −1 vs. 3.8 ± 0.6 mmol × l −1 and 3.0 ± 0.5 mmol × l −1 vs. 3.7 ± 0.6 mmol × l −1 , respectively; both p < 0.05). There was no difference in blood α-amino-N concentrations between GH or Ipamorelin treated animals. 3.4 N-loss and N-balance ( Fig. 2 ) Control animals were in positive N-balance (11.9 ± 2.0 mmol × 24 h −1 ). Fifty percent of the nitrogen was excreted as urea nitrogen, 10% as non-urea nitrogen in the urine, and 40% as faecal nitrogen. In Prednisolone treated rats total nitrogen excretion nearly doubled due to an increase in urinary urea nitrogen excretion, and N-balance became markedly negative (−9.8 ± 3.0 mmol × 24 h −1 vs. 11.9 ± 2.0 mmol × 24 h −1 , p < 0.01). Faecal nitrogen and urinary non-urea nitrogen excretions were identical. GH and Ipamorelin significantly reduced nitrogen loss compared to steroid treated animals (5.1 ± 1.5 mmol × 24 h −1 vs. −9.8 ± 3.0 mmol × 24 h −1 and 1.0 ± 0.6 mmol × 24 h −1 vs. −9.8 ± 3.0 mmol × 24 h −1 , respectively; p < 0.01). There was a significant difference in nitrogen excretion between GH and Ipamorelin treated rats ( p < 0.05). 3.5 The alanine stimulated capacity of urea-N synthesis rate (CUNS) ( Fig. 3 ) Prednisolone more than doubled CUNS compared to pair-fed animals (19.2 ± 1.1 μmol × min −1 × 100 g BW −1 vs. 8.1 ± 1.0 μmol × min −1 × 100 g BW −1 , p < 0.01). GH reduced the steroid-induced CUNS by 33% (12.9 ± 1.0 μmol × min −1 × 100 g BW −1 vs. 19.2 ± 1.1 μmol × min −1 × 100 g BW −1 , p < 0.01), whereas Ipamorelin reduced the steroid effect by 20% (15.3 ± 0.9 μmol × min −1 × 100 g BW −1 vs. 19.2 ± 1.1 μmol × min −1 × 100 g BW −1 , p < 0.05). There was no difference in CUNS between St+Gh and St+Ipamorelin. 3.6 Organ N-contents (mg N) ( Table 1 ) Changes in organ weight and N-contents were parallel and only the latter is given. Prednisolone treatment decreased organ N-contents of liver, heart and skeletal muscles, whereas kidney N remained unchanged compared to pair-fed controls. GH treatment normalized N-contents of all organs except the kidneys, which showed no change. Ipamorelin normalized N-contents of all organs except in the soleus muscle. 3.7 mRNA levels ( Figs. 4 and 5 ) Compared with pair-fed controls prednisolone increased mRNA abundance of the flux generating urea cycle enzyme carbamoyl phosphate synthetase (CPS) by 35% (62639 ± 3091 vs. 46470 ± 1150, p < 0.01) and increased that of the rate-limiting enzyme argininosuccinate synthetase (ASS) by 240% (39862 ± 3315 vs. 11694 ± 477, p < 0.01). GH (CPS: 44733 ± 2435 ASS: 15136 ± 1181) and Ipamorelin (CPS: 55809 ± 1808 ASS: 15916 ± 618) both normalized the expression of these enzymes. 3.8 Serum IGF-I ( Table 1 ) There was no difference in serum IGF-I between pair-fed controls and the steroid treated group. GH increased serum IGF-I by 50% compared to pair-fed controls ( p < 0.05), while there was no effect of Ipamorelin treatment compared to pair-fed controls. 4 Discussion Our main findings are that Ipamorelin and GH both counteracted steroid-induced catabolism, although the latter more efficiently. Both substances reduced the prednisolone-induced up-regulation of the in vivo rate of urea synthesis (CUNS) and they reduced the increased gene expression of key urea cycle enzymes. Simultaneously, body weight, whole-body N-balance, and organ N-contents all improved. The parallel changes in in vivo urea synthesis, urea enzyme gene expression, whole-body N-balance, and organ N-contents suggest that a significant part of the catabolic effects of prednisolone and the anabolic effects of GH and Ipamorelin probably involves regulation of urea synthesis on gene level. The anabolic actions of GH are partly due to stimulation of hepatic release of IGF-I, and we have earlier described a direct effect of IGF-I on hepatic urea synthesis and regulation of urea cycle enzymes in rats [25] . There are conflicting reports as to the effects of GH secretagogues on IGF-I levels [8,26–29] . Malmlöf et al., using similar steroid doses (5 mg × kg −1 × d −1 ) and Ipamorelin (0.4 or 1.6 mg × kg −1 × d −1 ), observed that Ipamorelin normalized GH secretion and IGF-I levels in steroid treated rats [29] .The observation that Ipamorelin did not increase IGF-I may indicate GH resistance in the liver as also observed in diabetic mice [28] . In a recent study Ipamorelin was found to counteract the decrease in bone formation by glucocorticoid, and in that study there was no difference in serum IGF-I between the groups studied, either [30] . We did not measure GH levels, though it might have enlightened this observation, as GH may stimulate local IGF-I production which may in turn exert autocrine and paracrine effects. There is no report of GH secretagogues having direct hepatic effects, and it has been shown that an intact pituitary is necessary for the effects of these GH releasing peptides [31] . Furthermore, glucocorticoids do not prevent Ipamorelin from stimulating GH release [29] . These findings may indicate that the observed effects of Ipamorelin on nitrogen conversion are mediated through Ipamorelin stimulated GH release from the pituitary gland. In the present study the metabolic effects of Ipamorelin were similar to previous studies with GH and IGF-I treatment of normal- and steroid treated rats [2,7,25,32] . In the present study, liver nitrogen content in the pair-fed controls is larger than in a previous study, whereas the other variables are similar [2] . We have no obvious explanation for this discrepancy. Thus, Ipamorelin metabolically acts in the same way as GH, although in the doses given here not as efficiently. Probably more marked stimulation of GH secretion is needed to elicit a full effect on muscle-N conservation and circulating IGF-I levels. Future, dose-finding studies may show whether this is achievable via higher Ipamorelin doses, or if desensitization of the pituitary to the Ipamorelin may be responsible [33] . However, the orexigenic effect of GH secretagogues may limit its usefulness. The induced experimental steroid catabolism was severe. In comparison with pair-fed controls it represented a 25% loss in body weight. The pair-feeding ensured that the weight loss was due to a reduction in nitrogen economy, i.e. metabolic events, rather than to reduced food intake. Likewise, the pair-feeding design ensured that the effects of GH and Ipamorelin on the steroid-induced decrease in N-balance and N-contents were due to metabolic rather than to dietary effects. The CUNS method standardizes urea synthesis with regard to substrate drive. Changes in CUNS, therefore, reflect regulatory non-substrate dependent changes in urea synthesis. This method has been validated in terms of correlation with established liver function tests [34,35] and has been applied in a series of experimental disease states [24,36] as well as in investigations of effects of various hormones on urea synthesis [3,37,38] . The decrease in urea synthesis caused by GH and Ipamorelin may be secondary to effects on protein synthesis and diversion of amino-N from the liver to muscles and/or it may be due to down-regulation of urea synthesis. The fall in blood amino acids, the increases in CUNS, and the increase in the expression of urea cycle enzymes indicate that regulation of liver function in terms of urea synthesis is of primary importance and contributes to nitrogen balance. In the present study we were able to compare in vivo metabolic events with determinations of expression of key genes for the urea cycle enzymes. Prednisolone increased gene expression of both the flux controlling feeder enzyme carbamoyl phosphate synthetase and of the rate-limiting enzyme, the argininosuccinate synthetase. The treatment of steroid catabolic animals with GH or Ipamorelin resulted in near-normal expression of both genes. The metabolic effects of prednisolone, GH and Ipamorelin thus corresponded qualitatively, and for the flux controlling enzyme on gene level also quantitatively, to the effects in vivo on the capacity of urea synthesis. This demonstrates that prednisolone as well as GH and Ipamorelin exerted effects on regulation of urea synthesis on gene level. The many steps between gene expression and physiological process are not necessarily of regulatory importance in the present context. In conclusion, we demonstrated that accelerated nitrogen wasting in the liver and other organs by prednisolone treatment was counteracted by treatment with either GH or its secretagogue Ipamorelin; at the doses given less efficiently by the latter. This may support a clinical rationale to treat steroid wasting with Ipamorelin. However, clinical studies are needed to confirm this. Acknowledgements The present study was supported by grants from Clinical Institute, Aarhus University; Danish Health Research Council; Novo Nordic Foundation for Growth and Regeneration; and “Savvæksejer Jeppe Juhl og hustru Ovita Juhls mindelegat”. Growth hormone and Ipamorelin were generously supplied by Novo Nordic, Gentofte, Denmark. The authors greatly appreciate the skilful technical assistance provided by Joan Didriksen, Lene Vestergård Jensen, Inger Schødt, Kirsten Nyborg, Kirsten Priisholm, and Bjørg Krog, and wish to thank Professor S. M. J. Morris for providing plasmids containing urea cycle enzyme cDNA sequences. References [1] B. Beaufrere F.F. Horber W.F. Schwenk H.M. Marsh D. Matthews J.E. Gerich M.W. Haymond Glucocorticosteroids increase leucine oxidation and impair leucine balance in humans Am. J. Physiol. 257 1989 E712 E721 [2] T. Grofte D.S. Jensen H. Gronbaek T. Wolthers S.A. Jensen N. Tygstrup H. Vilstrup Effects of growth hormone on steroid-induced increase in ability of urea synthesis and urea enzyme mRNA levels Am. J. Physiol. 275 1998 E79 E86 [3] I. Sigsgaard T. Almdal B.A. Hansen H. Vilstrup Dexamethasone increases the capacity of urea synthesis time dependently and reduces the body weight of rats Liver 8 1988 193 197 [4] K. Berneis R. Ninnis J. Girard B.M. Frey U. Keller Effects of insulin-like growth factor I combined with growth hormone on glucocorticoid-induced whole-body protein catabolism in man J. Clin. Endocrinol. Metab. 82 1997 2528 2534 [5] F.F. Horber M.W. Haymond Human growth hormone prevents the protein catabolic side effects of prednisone in humans J. Clin. Invest. 86 1990 265 272 [6] M. Oehri R. Ninnis J. Girard F.J. Frey U. Keller Effects of growth hormone and IGF-I on glucocorticoid-induced protein catabolism in humans Am. J. Physiol. 270 1996 E552 E558 [7] T. Wolthers T. Grofte J.O. Jorgensen H. Vilstrup Growth hormone prevents prednisolone-induced increase in functional hepatic nitrogen clearance in normal man J. Hepatol. 27 1997 789 795 [8] P.B. Johansen J. Nowak C. Skjaerbaek A. Flyvbjerg T.T. Andreassen M. Wilken H. Orskov Ipamorelin, a new growth-hormone-releasing peptide, induces longitudinal bone growth in rats Growth Horm. IGF Res. 9 1999 106 113 [9] K. Raun B.S. Hansen N.L. Johansen H. Thogersen K. Madsen M. Ankersen P.H. Andersen Ipamorelin, the first selective growth hormone secretagogue Eur. J. Endocrinol. 139 1998 552 561 [10] M. Ankersen N.L. Johansen K. Madsen A new series of highly potent growth hormone-releasing peptides derived from Ipamorelin J. Med. Chem. 41 1998 3699 3704 [11] T.K. Hansen M. Ankersen B.S. Hansen Novel orally active growth hormone secretagogues J. Med. Chem. 41 1998 3705 3714 [12] T. Grofte D.S. Jensen J. Greisen N. Tygstrup H. Vilstrup Growth hormone and insulin-like growth factor I counteracts established steroid catabolism in rats by effects on hepatic amino-N degradation J. Hepatol. 35 2001 700 706 [13] G. Ortoft H. Gronbaek H. Oxlund Growth hormone administration can improve growth in glucocorticoid-injected rats without affecting the lymphocytopenic effect of the glucocorticoid Growth Horm. IGF Res. 8 1998 251 264 [14] B.A. Hansen H. Vilstrup A method for determination of the capacity of urea synthesis in the rat Scand. J. Clin. Lab. Invest. 45 1985 315 320 [15] E.J. Henriksen R.E. Bourey K.J. Rodnick L. Koranyi M.A. Permutt J.O. Holloszy Glucose transporter protein content and glucose transport capacity in rat skeletal muscles Am. J. Physiol. 259 1990 E593 E598 [16] S. Cerdan C.J. Lusty K.N. Davis J.A. Jacobsohn J.R. Williamson Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I J. Biol. Chem. 259 1984 323 331 [17] P. Mcintyre J.F. Mercer M.G. Peterson P. Hudson N. Hoogenraad Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli . Molecular cloning of rat ornithine transcarbamylase Eur. J. Biochem. 1984 183 187 [18] L.C. Surh S.M. Morris W.E. O’Brien A.L. Beaudet Nucleotide sequence of the cDNA encoding the rat argininosuccinate synthetase Nucleic Acids Res. 16 1988 9352 [19] Y. Amaya T. Matsubasa M. Takiguchi Amino acid sequence of rat argininosuccinate lyase deduced from cDNA J. Biochem. 103 1988 177 181 [20] G.J. Dizikes E.B. Spector S.D. Cederbaum Cloning of rat liver arginase cDNA and elucidation of regulation of arginase gene expression in H4 rat hepatoma cells Somat Cell Mol. Genet. 12 1986 375 384 [21] J.K. Fawcett J.E. Scott A rapid and precise method for the determination of urea J. Clin. Pathol. 13 1960 156 159 [22] J.F. Goodwin Spectrofotometric quantitation of plasma and urinary amino-nitrogen with flourid St. Meth. Clin. Chem. 6 1970 89 98 [23] T.P. Almdal H. Vilstrup Effects of streptozotocin-induced diabetes and diet on nitrogen loss from organs and on the capacity of urea synthesis in rats Diabetologia 30 1987 952 956 [24] T.P. Almdal K.F. Petersen B.A. Hansen H. Vilstrup Increased capacity of urea synthesis in streptozotocin diabetes in rats Diabetologia 29 1986 812 816 [25] T. Grofte T. Wolthers S.A. Jensen Effects of growth hormone and insulin-like growth factor I singly and in combination on in vivo capacity of urea synthesis, gene expression of urea cycle enzymes, and organ nitrogen contents in rats Hepatology 25 1997 964 969 [26] B.S. Hansen K. Raun K.K. Nielsen Pharmacological characterisation of a new oral GH secretagogue, NN703 Eur. J. Endocrinol. 141 1999 180 189 [27] T. Jacks R. Smith F. Judith MK-0677, a potent, novel, orally active growth hormone (GH) secretagogue: GH, insulin-like growth factor I, and other hormonal responses in beagles Endocrinology 137 1996 5284 5289 [28] P.B. Johansen Y. Segev D. Landau M. Phillip A. Flyvbjerg Growth hormone (GH) hypersecretion and GH receptor resistance in streptozotocin diabetic mice in response to a GH secretagogue Exp. Diabesity Res. 4 2003 73 81 [29] K. Malmlof P.B. Johansen P.M. Haahr M. Wilken H. Oxlund Methylprednisolone does not inhibit the release of growth hormone after intravenous injection of a novel growth hormone secretagogue in rats Growth Horm. IGF Res. 9 1999 445 450 [30] N.B. Andersen K. Malmlof P.B. Johansen T.T. Andreassen G. Ortoft H. Oxlund The growth hormone secretagogue Ipamorelin counteracts glucocorticoid-induced decrease in bone formation of adult rats Growth Horm. IGF Res. 11 2001 266 272 [31] K.D. Schleim T. Jacks P. Cunningham Increases in circulating insulin-like growth factor I levels by the oral growth hormone secretagogue MK-0677 in the beagle are dependent upon pituitary mediation Endocrinology 140 1999 1552 1558 [32] T. Wolthers T. Grofte N. Moller H. Vilstrup J.O. Jorgensen Effects of long-term growth hormone (GH) and triiodothyronine (T3) administration on functional hepatic nitrogen clearance in normal man J. Hepatol. 24 1996 313 319 [33] G.J. Hickey T.M. Jacks K.D. Schleim Repeat administration of the GH secretagogue MK-0677 increases and maintains elevated IGF-I levels in beagles J. Endocrinol. 152 1997 183 192 [34] B.A. Hansen H.E. Poulsen The capacity of urea-N synthesis as a quantitative measure of the liver mass in rats J. Hepatol. 2 1986 468 474 [35] H. Vilstrup On urea synthesis–regulation in vivo Dan. Med. Bull. 36 1989 415 429 [36] K. Bjerrum H. Vilstrup T.P. Almdal K.L. Ostergaard No effect of bicarbonate-induced alkalosis on urea synthesis in normal man Scand. J. Clin. Lab. Invest. 50 1990 137 141 [37] B.A. Hansen B. Krog H. Vilstrup Insulin and glucose decreases the capacity of urea-N synthesis in the rat Scand. J. Clin. Lab. Invest. 46 1986 599 603 [38] K.F. Petersen B.A. Hansen H. Vilstrup Time dependent stimulating effect of glucagon on the capacity of urea-N synthesis in rats Horm. Metab. Res. 19 1987 53 56