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Dev Growth Differ
2023 Jan 01;651:6-15. doi: 10.1111/dgd.12833.
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Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis.
Konno N
.
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Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10-500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.
Figure 1. Detection of mRNA-expression levels of genes encoding urea cycle enzymes and urea excretion in premetamorphic tadpoles. (A) Tissue-expression patterns of carbamoyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC) mRNAs in stage-52 tadpole (21 dpf), as detected by RT-PCR. (B and C) expression levels of CPSI and OTC mRNAs in whole tadpoles during premetamorphosis, as detected using RT-PCR. (D) Expression level of CPSI mRNA during premetamorphosis, as detected using quantitative real-time RT-PCR. The values shown represent the expression levels normalized to that of the internal standard gene EF1α and are presented as the mean ± standard error of the mean (SEM) for five tadpoles at each stage. The different letters shown in the plots indicate significant differences at p < .05 (based on one-way analysis of variance with Tukey's multiple-comparison test). (E) Change in urea excretion during premetamorphosis. The values shown represent the mean ± SEM of 6–12 tadpoles at each stage. The different letters shown the in plots indicate significant differences at p < .05 (based on one-way analysis of variance with Tukey's multiple-comparison test).
Figure 2. Contributions of glucocorticoids to ureagenesis in premetamorphic tadpoles. (a) Effect of Dex exposure on hepatic carbamoyl phosphate synthetase I (CPSI) mRNA expression in stage-46/47 tadpoles. The values shown represent the mean ± SEM of seven tadpoles at stage 46/47. The different letters shown in the plots indicate significant differences at p < .05 (based on one-way analysis of variance with Tukey's multiple-comparison test). (b) Premetamorphic glucocorticoid receptor (GR) mRNA expression, as determined by quantitative real-time RT-PCR. The values shown represent the mean ± SEM of four tadpoles at each stage. The different letters shown in the plots indicate significant differences at p < .05 (based on one-way analysis of variance with Tukey's multiple-comparison test).
Figure 3. Expression levels of genes encoding gluconeogenic enzymes during premetamorphosis. The gene-expression levels of five gluconeogenic enzymes (a, G6Pase α; b, G6Pase β; c, PEPCK1; d, PEPCK2; e, FBP1) were analyzed using quantitative real-time RT-PCR. The values shown represent the expression levels normalized to that of the internal standard gene EF1α and are presented as the mean ± SEM of five tadpoles at each stage. The different letters shown in the plots indicate significant differences at p < .05 (based on one-way analysis of variance with Tukey's multiple-comparison test).
Figure 4. Relationship between ureagenesis and gluconeogenesis during Xenopus development. (a) Predicted metabolic processes of ureagenesis and gluconeogenesis in premetamorphic Xenopus tadpoles. CPSI and OTC mRNA expression were detected for the first time at stage 47, concurrently with the first detection of urea excretion. Dex exposure upregulated CPSI mRNA expression. In addition, the expression levels of genes encoding gluconeogenic enzymes simultaneously increased. (b) Relationship between hormone levels and metabolism during X. laevis development. Transient GR mRNA expression and a corticosterone surge (Kloas et al., 1997) coincided with increased expression of genes encoding urea cycle enzymes and gluconeogenic enzymes (around stage 47). The data related to corticosterone and thyroid hormones (T3 and T4) levels during Xenopus development were originally published by Kloas et al. (1997) and Leloup (1977), respectively. Arg, arginase; ASL, argininosuccinate lyase; AsuS, argininosuccinate synthase; CORT, corticosterone; CPSI, carbamoyl phosphate synthetase 1; FBP1, fructose 1,6-bisphosphatase; G6Pase, glucose 6-phosphatase; GR, glucocorticoid receptor; OTC, ornithine transcarbamylase; PEPCK, phosphoenolpyruvate carboxykinase; T3, triiodothyronine; T4, thyroxine
Figure S1. Change in ammonia excretion and the ratios of ammonia and urea excretion for total nitrogen excretion during premetamorphosis. (A) Ammonia excretion in premetamorphic tadpoles. The values for each stage are presented as mean ± SEM for 6–12 tadpoles. Different letters in plots indicate differences that are statistically significant at p < 0.05 (one-way analysis of variance with Tukey’s multiple comparison test). (B) The ratios of ammonia and urea excretion for total nitrogen excretion in the premetamorphic tadpoles. The values for each stage are presented as mean ± SEM for 6–12 tadpoles. Results are analyzed by one-way analysis of variance with Tukey’s multiple comparison test. Different letters in plots indicate significant differences at p < 0.05.
