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