T3 treatment reduced the food intake of Rana omeimontis tadpoles (Figure 1B). After 3–4 d of treatment, these tadpoles had decreased weight (Figure 1C), accelerated hind limb development and tail absorption (Figure 1D–E), and broadened oral disk width (Figure 1F). In contrast to the increased consumption of hepatic resource in starved pro-metamorphic tadpoles , T3-treated tadpoles had a liver size similar to the control group despite their reduced food intake (Figure 1F and Additional file 2: Figure S2). When food was not provided during treatment, T3-treated tadpoles had larger livers than the control group (Figure 1F and Additional file 2: Figure S2).
Dramatic metabolic reorganization during onset of metamorphic climax
Metamorphosis from pro-metamorphic to metamorphic stages was associated with dramatic metabolic adjustments. The variation of liver and tail metabolomes divided tadpoles into pro-metamorphic (stages 36 and 41) and metamorphic groups (stages 43 and 44) along the first primary component (PC1, accounting for 31.1% of the total variance) of PCA (Figure 2A–B and Additional file 2: Figure S3; see detailed metabolomic data in Tables S1–S4). At transcriptional level, KEGG enrichment analyses were conducted for DEGs between T3-treated and control groups (Additional file 2: Figure S1). Metabolic pathways accounted for the largest proportion of the top 30 significantly enriched items (14/30 for the liver and 11/30 for the tail; Figure 2C–D; Table S5–S6).
Lipid metabolism in the liver during metamorphic climax
With the progression of metamorphosis from stage 36 to stage 44, four free fatty acids (FFAs; Δ18:1, Δ18:3, Δ16:0, and Δ16:1) and two aryl-carnitines (Δ18:0-carnitine and Δ10:0-carnitine; active form of FFAs) decreased and increased in content, respectively (p < 0.05, one-way ANOVA; Figure 3A–B). Levels of other FFAs and aryl-carnitines were unchanged. Three FFAs (Δ18:1, Δ18:2, and Δ16:0) and two acyl-carnitines (Δ16:0-carnitine and Δ18:0-carnitine) decreased and increased in content, respectively, in T3-treated groups (Figure 3C). Other FFAs or acyl-carnitines were unaffected. At the transcriptional level, T3-treated tadpoles showed upregulated lipogenesis (diacylglycerol/DAG O-acyltransferases), but downregulated lipolysis (hepatic triacylglycerol/TAG lipase and DAG lipase), fatty acid transport (fatty acid binding protein and long-chain fatty acid transport protein), FFA β-oxidation (acyl-CoA dehydrogenase and trifunctional protein), and other types of FFA oxidations such as fatty aldehyde dehydrogenase and fatty acid 2-hydroxylase (Figure 3D). Correspondingly, cholesterol synthesis, the downstream pathway of FFA oxidation, was also downregulated at the transcriptional level (Figure 3D). Bile acid and steroid hormone metabolism, the catabolic routes of cholesterol, were also downregulated (Additional file 2: Figure S4). Histological sections indicated that T3-treated tadpoles contained more hepatic fat (larger vacuoles in H&E staining and larger red area in red oil staining) than the control group (Figure 3E). Taken together, these results suggest that hepatic fat consumption was reduced after the onset of metamorphic climax, and the fatty acid flux in the liver was encouraged to flow into TAG synthesis rather than degradation and sterol synthesis (Figure 3F). The liver of metamorphic tadpoles showed decreased expression of peroxisome proliferators-activated receptor alpha (PPARα) (Additional file 2: Figure S5).
Lipid metabolism in the tail during metamorphic climax
Metamorphosis from pro-metamorphic stages (stages 36 and 41) to metamorphic climax (stage 43) was accompanied by a dramatic increase of most unsaturated FFAs, acyl-carnitines, and MAG (Figure 4A). T3 treatment partly reproduced these metabolic changes in pro-metamorphic tadpoles by inducing the levels of Δ16:1, Δ17:0, Δ18:1, Δ20:4, and Δ18:0-carnitine (Figure 4B). At the transcriptional level, T3-treated tadpoles showed upregulated glycerolipid synthesis (DAG/MAG O-acyltransferase), phospholipid degradation (phospholipases), and fatty acid elongation and desaturation (fatty acid desaturases and elongation of long chain fatty acid proteins), but downregulated FFA β-oxidation (2-ketoacyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase) (Figure 4C–D). These results suggest an accelerated degradation of phospholipids during metamorphic climax. The resulting FFAs flux was mainly diverted to synthesis of glycerolipid and long-chain unsaturated fatty acid, rather than to further catabolism (Figure 4E). T3-treated tadpoles had increased transcription of PPARα, PPARβ, and PPARγ in their tail (Additional file 2: Figure S5). Their tail also showed decreased transcription of adiponectin, a secretory metabolic regulator, while the transcription of adiponectin receptors was upregulated (Additional file 2: Figure S5).
The tails of metamorphic tadpoles showed accumulation of prostaglandins (PGs) and hydroperoxyeicosatetraenoic acid (HETE) (Figure 4F) that are derivatives of unsaturated FFAs. T3 treatment upregulated arachidonic acid metabolism (PG synthases and cytochrome P450) (Figure 4G–I and Additional file 2: Figure S6), which was responsible for synthesizing these derivatives. These results suggest increased synthesis of functional FFA derivatives in the tail during metamorphic climax.
Carbohydrate metabolism in the liver during metamorphic climax
Metamorphic tadpoles (stages 43 and 44) had decreased levels of hepatic disaccharides and trisaccharides (the major forms of soluble sugar in the liver of R. omeimontis tadpoles such as maltose and maltotriose) (Figure 5A). Consistent with that, T3-treated tadpoles had downregulated transcription of glycogen debranching enzyme and α-amylase in their liver (Figure 5B). This suggests reduced mobilization of glycogen through hydrolyzation. Metamorphic tadpoles showed increased transcription of glycogen phosphorylase and an increased level of glucose 1-phosphate (Figure 5A–B), suggesting that glycogen mobilization was maintained through the phosphorylation route during metamorphic climax. Phosphoglucomutase was the critical enzyme diverting glucose 1-phosphate to glycolysis and PPP by converting it to glucose 6-phosphate. In the liver of metamorphic tadpoles, its downregulation and decreased transcription of enzymes involved in glycolytic enzymes (phosphoglucose isomerase and fructose-biphosphate aldolase) suggested reduced metabolic fluxes throughout glycolysis (Figure 5B–C), even though the level of glycolytic intermediates (hexose 6-phosphates and fructose 1,6-biphosphate) was maintained (Figure 5A). This was consistent with the increased transcription of phosphoenolpyruvate carboxykinases (PECKs) (Figure 5B), the critical enzymes of gluconeogenesis. Similarly, metamorphic tadpoles had decreased levels of PPP intermediates (gluconate 6-phosphate and ribulose 5-phosphate) and downregulated transcription of ribulose-phosphate 3-epimerase (Figure 5B–C), suggesting reduced metabolic flux throughout PPP during metamorphic climax. UDP-glucose 6-dehydrogenase is responsible for converting glucose 1-phosphate to UDP-glucuronate. Its upregulated transcription and increased levels of UDP-glucuronate and related metabolites in metamorphic tadpoles suggests that glucuronate interconversion was enhanced and likely responsible for the increased metabolic flux from glycogen to glucose 1-phosphate (Figure 5A–C).
Carbohydrate metabolism in the tail during metamorphic climax
Because the two pro-metamorphic stages (stages 36 and 41) differed in their tail profiles of glycolytic metabolites (Figure 5D), our analyses focused on the differences between stage 41 and 43 to highlight the metabolic changes associated with the onset of metamorphic climax. The levels of disaccharides and trisaccharides (maltotriose and maltopentaose) in their tail was maintained when metamorphosis proceeded from stage 41 to 43 (Figure 5D). Although the transcription of glycogen debranching enzyme was downregulated in metamorphic tadpoles, their transcription of α-amylase was upregulated (Figure 5E). These results suggest that glycogen mobilization in the form of disaccharides and trisaccharides was maintained. The decreased transcription of glycogen phosphorylase suggests reduced mobilization of glycogen through phosphorylation (Figure 5E). The increased transcription of glycogen synthase and decreased glycogen synthase kinase suggests that glycogen synthesis was suppressed during metamorphic climax (Figure 5E). Metamorphic tadpoles (stage 43) maintained higher levels of hexose phosphates (fructose 1-phosphate and fructose 6-phosphate) than pro-metamorphic tadpoles (stage 41) (Figure 5D). This was consistent with the increased transcription of hexokinases and glucokinase in T3-treated tadpoles (Figure 5E), suggesting increased metabolic flux from soluble sugar (glycogenolysis and tissue apoptosis) to hexose phosphates. This carbohydrate flux was not likely diverted into glycolysis, as the transcription of glycolytic enzymes (triosephosphate isomerase) was downregulated by T3 treatment (Figure 5E). In contrast, PPP and glucuronate and glucosamine metabolism were likely encouraged during metamorphic climax, as metamorphic tadpoles maintained increased levels of intermediates in PPP and glucose derivates (glucuronate and glucosamine) (Figure 5D and 5F), as well as increased transcription of related enzymes (Figure 5E). These results suggest that the carbohydrate flux in the tail was preferentially allocated to metabolic shunts associated with biosynthesis, rather than energy production (Figure 5G).
Protein and amino acid metabolism during metamorphic climax
Amino acids and dipeptides increased in the liver and tail after the onset of metamorphic climax (Figure 6A). T3-treatment induced increased transcription of metallopeptidases, dipeptidases, and cathepsins in the tail, but not in the liver (Figure 6B), while increased transcription of amino acid transporters was observed in both the liver and tail (Figure 6C). These results suggest accelerated protein degradation in the tail and increased amino acid flux from the tail to liver during metamorphic climax.
In the liver, metamorphic tadpoles showed decreased transcription of aminoacyl-tRNA synthetases and ribosomal components but increased transcription of aminotransferases and enzymes of urea cycle (carbamoyl-phosphate synthase and argininosuccinate lyase) (Figure 6C–D). This suggested that amino acid catabolism increased in the liver, rather than protein synthesis, during metamorphic climax (Figure 6E).
In the tail, the transcription of aminoacyl-tRNA synthetase, ribosomal components, and most aminotransferases was downregulated (Figure 6C–D), suggesting simultaneous suppression of amino acid catabolism and protein synthesis during metamorphic climax (Figure 6E). T3 treatment increased the transcription of glutamine synthetase in the tail (Figure 6C), and the levels of glutamine increased in both tail and liver of metamorphic tadpoles (Additional file 2: Figure S7). This suggested that tail ammonia was recycled in the form of glutamine during metamorphic climax (Figure 6E).
TCA cycle and oxidative phosphorylation during metamorphic climax
TCA cycle and oxidative phosphorylation are common downstream processes of lipid, carbohydrate, and amino acid catabolism. Metamorphic tadpoles showed an overall upregulated transcription of these two pathways in their liver, but downregulated transcription in their tail (Additional file 2: Figure S8 A–C). In the tail, Ca2+-ATPases and muscle creatine kinases, the primary ATP consumers in muscle, were also downregulated, while the transcription of uncoupling protein (UCP), which diverts proton gradient away from energy production, was upregulated (Additional file 2: Figure S8 D).