Remarkable metabolic reorganization and altered metabolic requirements in frog metamorphic climax

Abstract

Background: Metamorphic climax is the crucial stage of amphibian metamorphosis responsible for the morphological and functional changes necessary for transition to a terrestrial habitat. This developmental period is sensitive to environmental changes and pollution. Understanding its metabolic basis and requirements is significant for ecological and toxicological research. Rana omeimontis tadpoles are a useful model for investigating this stage as their liver is involved in both metabolic regulation and fat storage.

Results: We used a combined approach of transcriptomics and metabolomics to study the metabolic reorganization during natural and T3-driven metamorphic climax in the liver and tail of Rana omeimontis tadpoles. The metabolic flux from the apoptotic tail replaced hepatic fat storage as metabolic fuel, resulting in increased hepatic amino acid and fat levels. In the liver, amino acid catabolism (transamination and urea cycle) was upregulated along with energy metabolism (TCA cycle and oxidative phosphorylation), while the carbohydrate and lipid catabolism (glycolysis, pentose phosphate pathway (PPP), and β-oxidation) decreased. The hepatic glycogen phosphorylation and gluconeogenesis were upregulated, and the carbohydrate flux was used for synthesis of glycan units (e.g., UDP-glucuronate). In the tail, glycolysis, β-oxidation, and transamination were all downregulated, accompanied by synchronous downregulation of energy production and consumption. Glycogenolysis was maintained in the tail, and the carbohydrate flux likely flowed into both PPP and the synthesis of glycan units (e.g., UDP-glucuronate and UDP-glucosamine). Fatty acid elongation and desaturation, as well as the synthesis of bioactive lipid (e.g., prostaglandins) were encouraged in the tail during metamorphic climax. Protein synthesis was downregulated in both the liver and tail. The significance of these metabolic adjustments and their potential regulation mechanism are discussed.

Conclusion: The energic strategy and anabolic requirements during metamorphic climax were revealed at the molecular level. Amino acid made an increased contribution to energy metabolism during metamorphic climax. Carbohydrate anabolism was essential for the body construction of the froglets. The tail was critical in anabolism including synthesizing bioactive metabolites. These findings increase our understanding of amphibian metamorphosis and provide background information for ecological, evolutionary, conservation, and developmental studies of amphibians.

Keywords: Amphibian; Metabolic reorganization; Metabolic switch; Metamorphosis.

Fig. 1

Experimental design and T3-driven metamorphic climax. a Experimental design. b–g T3-induced morphological and physiological changes in pro-metamorphic R. omeimontis tadpoles (stages 30–31). T3-treated tadpoles had reduced food intake (b), reduced body weight (c), accelerated development of hind limbs (d), shortened tail (e), broadened oral disk width (f–g), and reduced mobilization of hepatic resources (f–g; reflected by the liver size and morphology). Food intake was reflected by the residual content of spirulina powder in the water; the higher content of the spirulina powder, the darker the green color of the water. p < 0.001

Fig. 2

Dramatic metabolic reorganization during metamorphic climax. a and b Scatter plots of PCAs based on liver (a) and tail (b) metabolomes of tadpoles at different Gosner stages (n = 6 for each organ at each stage). c and d Top 30 significantly enriched KEGG pathway based on liver (c) and tail (d) DEGs between T3-treated and control tadpoles. The pathway categories were adapted from the KEGG pathway database. The cover rate is the ratio between number of genes enriched in a pathway and the total number of genes in this pathway

Fig. 3

Reorganization of lipid metabolism in the liver during metamorphic climax. a–b Free fatty acids (FAAs) and acylcarnitines varied (p < 0.05, one-way ANOVA) during natural metamorphosis. Different letters denote significant differences between groups (p < 0.05), as shown by the Student–Newman–Keuls post hoc test after one-way ANOVA. c FFAs and acylcarnitines differed in content between control and T3-treated groups. Each box represents a mean ± SE; *, p < 0.05. d Transcriptional changes of genes involved in lipid metabolism in the liver after T3-treatment; a positive log-transformed fold change value means upregulation in T3-treated group, and vice versa; *, p < 0.05. e Histological sections of the liver. Triacylglycerol (TAG) is the major form of hepatic fat storage in the liver and accounts for the red color in Oil Red O (ORO) staining. f Network presenting the adjustments on lipid metabolism in the liver. Metabolic fluxes are presented as arrows between items. Items and arrows with blue, red, cyan, and black colors indicate downregulated/decreased, upregulated/increased, unchanged, and undetected, respectively; and similarly hereinafter

Fig. 4

Reorganization of lipid metabolism in the tail during metamorphic climax. a Heatmap showing the variation of fatty acids during natural metamorphosis; #, p < 0.05 (one-way ANOVA). b FFAs and acylcarnitines differed in content between control and T3-treated groups; *, p < 0.05. c–d Transcriptional changes of genes involved in lipid metabolism after T3-treatment; *, p < 0.05. e Network denotes adjustments on lipid metabolism in the tail. f FFA derivatives/bioactive lipids varied in content (p < 0.05, one-way ANOVA) during natural metamorphosis (PG, prostaglandin; HETE, hydroperoxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid). Different letters denote significant differences between groups (p < 0.05), as determined by the Student–Newman–Keuls post hoc test after one-way ANOVA. g Transcriptional changes of critical enzymes involved in prostaglandin and leukotrienes biosynthesis. *, p < 0.05; **, p < 0.01. (H) Differently expressed cytochrome P450 genes involved in arachidonic acid metabolism (p < 0.05). (I) Network denotes the upregulated synthesis of FFA derivatives

Fig. 5

Reorganization of carbohydrate metabolism during metamorphic climax. a–c Carbohydrate metabolism in the liver. a Heatmap showing the variation of metabolites of glycol-metabolism when metamorphosis proceeds from stage 36 to stage 44, #, p < 0.05 (one-way ANOVA). b Transcriptional changes of genes involved in carbohydrate metabolism in the liver after T3-treatment; a positive log-transformed fold change value means upregulation in T3-treated group, and vice versa; *, p < 0.05. c Network denotes adjustments on lipid metabolism in the liver. (D–G) Carbohydrate metabolism in the tail. d Heatmap showing the variation of metabolites of glycol-metabolism when metamorphosis proceeds from stage 36 to stage 43, #, p < 0.05 (one-way ANOVA). e Transcriptional changes of genes involved in carbohydrate metabolism in the tail after T3-treatment; a positive log-transformed fold change value means upregulation in T3-treated group, and vice versa; *, p < 0.05. f Variation of metabolites in the pentose phosphate pathway (PPP) after T3-treatment. Each box represents the mean ± SE; *, p < 0.05. g Network denotes adjustments on lipid metabolism in the tail

Fig. 6

Amino acid metabolism during metamorphic climax. a Variation of amino acids and dipeptide during natural and T3-driven metamorphic climax. b–c Transcriptional changes of genes involved in amino acid metabolism in the liver after T3-treatment; a positive log-transformed fold change value means upregulation in the T3-treated group, and vice versa; *, p < 0.05. d Heatmap showing the variation of ribosomal components (FPKM > 50) during natural metamorphosis. e Network denotes adjustments on amino acid metabolism

Fig. 7

Variation of metabolic fluxes during metamorphic climax. Items and arrows with blue, red, and cyan colors mean downregulated/decreased, upregulated/increased, and unchanged, respectively. The thickness of arrows is uniform, and it does not indicate the level of metabolic flux. Arrows in dashed lines indicates deduced adjustment on this metabolic pathway