Short-term fasting influences glucose and lipid metabolism in zebrafish. WT zebrafish at 3 mpf were fasted for 0 h to 80 h after the last feeding. (A,B) The serum glucose levels (A) and total lipid contents in the whole fish (B) were evaluated (n = 3 replicates, 3 fish/replicate). (C–F) Relative expression levels of glycolysis-related genes (gck and pklr) (C), gluconeogenesis-related genes (pck1 and fbp1a) (D), lipolysis-related genes (ppara and pgc1a) (E), and lipogenesis-related genes (pparg and fasn) (F) in the liver were determined (n ≥ 4/group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no statistical significance.

1,25(OH)₂D₃ generation in zebrafish was impaired under short-term fasting condition. (A–C). The gene expression of cyp2r1 (A), cyp27b1 (B) and cyp24a1 (C) in zebrafish liver was assessed after fasting for 24 h or 48 h, compared to the control group (1 h postprandial) (n = 4/group). (D) The serum 1,25(OH)₂D₃ concentrations were determined (n = 3 replicates, 4~5 fish/replicate). (E,F) Gene expression levels of vdra (E) and vdrb (F) in the liver (n = 4/group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no statistical significance.

VD regulates glucose metabolism in zebrafish under short-term fasting conditions. (A–C) After the feeding trial, zebrafish fed with VD₃ or non-VD₃ diet were fasted for 24 h before sampling. The gene expression of insra, insrb, gcgra, gcgrb (A) and gck, pklr, pck1, fbp1a (B) in the liver was measured (n = 4/group). Serum glucose levels were determined (C) (n = 3~4 replicates, 4~5 fish/replicate). (D–F) WT and cyp2r1^−/− zebrafish at 3 mpf were fasted for 24 h before sampling. The gene expression of insra, insrb, gcgra, gcgrb (D) and gck, pck1 (E) in the liver was analyzed (n = 4/genotype). Serum glucose levels were determined (F) (n = 4 replicates, 3 fish/replicate). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no statistical significance.

VD regulates lipid metabolism in zebrafish under short-term fasting conditions. (A–C) After the last feeding, zebrafish fed with VD₃ or non-VD₃ diet were fasted for 24 h before sampling. The gene expression of ppara, pgc1a, pparg, fasn (A) (n = 4/group), and FFA absorption-related genes cd36, slc27a2a (B) (n = 5/group) in the liver were determined. The gene expressions of ppara, pgc1a, fasn in the dorsal muscle were analyzed (C) (n = 6/group). (D–F) WT and cyp2r1^−/− zebrafish at 3 mpf were fasted for 24 h before sampling. The gene expressions of ppara, pgc1a, pparg, fasn (D) (n = 4/genotype), and cd36, slc27a2a (E) (n = 4/genotype) in the liver were determined. The gene expressions of ppara, pgc1a, fasn in the dorsal muscle were determined (F) (n = 4/genotype). * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no statistical significance.

VD₃ promotes the synthesis and processing of GLP-1 in the gut under short-term fasting conditions. (A–D) Zebrafish were fed with or without VD₃ for a month and fasted for 24 h. The gene expression of gip, gipr, pyya, pyyb, sglt (A) and gcga, gcgb, pcsk1, pcsk2 (B) in the gut was analyzed (n = 4/group). GLP-1 levels in the serum (C) and the intestine (D) were measured (n = 3 replicates, 4~5 fish/replicate). (E–H) WT and cyp2r1^−/− zebrafish at 3 mpf fasted for 24 h. The gene expression of gip, gipr, pyya, pyyb, sglt (E), and gcga, gcgb, pcsk1, pcsk2 (F) in the intestine was analyzed (n = 5/genotype). GLP-1 levels in the serum (G) (n = 4 replicates, 3 fish/replicate) and the intestine (H) (n = 3/ genotype) were measured. * p < 0.05, ** p < 0.01, ns: no statistical significance.

Interaction between VD3 and gut microbiota under short-term fasting conditions. (A–F) Zebrafish were treated with an antibiotic cocktail (Abx) while fed a VD3 or non-VD3 diet for one month. The serum glucose levels (A) and serum GLP-1 levels (B) were assayed (n = 3 replicates, 4~5 fish/replicate). The expression levels of gcga (C), gcgb (D), pcsk1 (E) and pcsk2 (F) in the intestine were analyzed (n = 4/group). (G) GF zebrafish at 3 dpf were incubated with NaA, NaP, or NaB (30 mM) for two days. The gene expression of pcsk1 was determined (n = 5 replicates, 10 larvae/replicate). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # p < 0.05, ## p < 0.01, ### p < 0.001, ns: no statistical significance.