The knockout of glut2 in zebrafish. (A) Transcriptional expression analysis of glut2 in different tissues (n = 6). (B) Sequence comparison of glut2 alleles in the control and two glut2 knockout zebrafish lines. (C) Schematic representation of glut2 full-length putative peptide from the control and two glut2 knockout fish lines. aa, amino acids. (D) The transcriptional expression of glut2 in 5 dpf control and glut2^−/− fish in the F3 population of both lines 1 and 2 (n = 3). (E) The hatching rate analysis of the control (glut2^+/+), glut2^+/−, and glut2^−/− fish from 0–3 dpf in the F3 population of line 2 (n = 6). (F) Analysis of the control, glut2^+/−, and glut2^−/− survival rates from 6 to 21 dpf in line 2 F3 population (236 fish were genotyped for analysis). (G–I) Representative images of the overall morphology of the control, glut2^+/−, and glut2^−/− fish at 30 dpf. (J, K) Body weight and body length of the control (n = 26), glut2^+/− (n = 53), and glut2^−/− (n = 26) fish at 30 dpf. A two-tailed unpaired t-test was used to detect significance. ****P < 0.0001. ns, no significance.

glut2-deletion results in growth retardation in MZglut2 fish. (A) The hatching rate analysis of the control and MZglut2 fish from 0 to 3 dpf (n = 6). (B) Survival rate analysis of the control and MZglut2 fish from 6–21 dpf (200 fish of each genotype were used for analysis). (C, D) Representative images of the overall morphology of the control and MZglut2 fish at 3 mpf. (E, F) Body weight and body length of the control and MZglut2 fish at 3 mpf (n = 21). A two-tailed unpaired t-test was used to detect significance. ****P < 0.0001.

glut2-deletion results in impaired glucose uptake in MZglut2 fish. (A) Glucose content in the blood of the control and MZglut2 fish (n = 12). (B) Glucose content in the muscle of the control and MZglut2 fish (n = 8). (C) Glucose content in the liver of the control and MZglut2 fish (n = 8). (D) The hepatic glucose uptake levels (2-DG) of the control and MZglut2 fish (n = 20). (E) Blood glucose levels of the control and MZglut2 fish after the glucose tolerance test for 20, 40, 60, 80, 100, and 120 min (n = 6). (F) Blood glucose levels of the control and MZglut2 fish after being fed a high carbohydrate diet for 0, 1, 2, 3, 4.5, 6, and 8 h (n = 6). (G) Transcriptional expression of glut2, glut1, glut3, glut5, glut8, glut9, glut12, and sglt1 in the intestine of the control and MZglut2 fish after fed high carbohydrate diet at 2 h (n = 6). (H) Transcriptional expression of glut2, glut1, glut3, glut5, glut8, glut9, glut12, and sglt1 in the liver of the control and MZglut2 fish after fed high carbohydrate diet at 2 h (n = 6). A two-tailed unpaired t-test was used to detect significance. ****P < 0.0001. ***P < 0.001. **P < 0.01. *P < 0.05. ns, no significance.

glut2-deletion results in impaired insulin-mediated anabolic metabolism in MZglut2 fish. (A) Representative images of β-cells in the control and MZglut2 larvae at 5 dpf. (B) The statistical analysis of β-cells in the control (n = 19) and MZglut2 (n = 18) larvae at 5 dpf. (C) Representative images of β-cells in DMSO- and Fisetin-treated larvae at 5 dpf. (D) The statistical analysis of β-cells in DMSO- and Fisetin-treated larvae at 5 dpf (n = 16). (E) Representative images of EdU stained β-cells in the control fish at 5 dpf. Red signal-positive β-cells are indicated by arrows (n = 8). (F) Representative images of EdU staining β-cells in MZglut2 larvae at 5 dpf (n = 8). (G) WISH using the insulin probe to the control and MZglut2 larvae at 5 dpf, with the green arrow representing the signal area. (H) The transcriptional expression levels of insra, insrb, chrebp, srebf1, and srebf2 in the liver of the control and MZglut2 fish at 3 mpf (n = 6). (I) The transcriptional expression levels of aclya, fasn, acc, fads2, scd, dgat1a, and dgat2 in the liver of the control and MZglut2 fish at 3 mpf (n = 6). (J) The transcriptional expression levels of atgl, lpl, pparab, cpt1aa, cpt1ab, and acox3 in the liver of the control and MZglut2 fish at 3 mpf (n = 6). (K) The quantification of carnitine metabolites based on the observations of metabolomics in the liver of control and MZglut2 fish at 3 mpf (n = 6). (L) The measurement of the lipid content of the control (n = 9) and MZglut2 (n = 8) fish at 3 mpf. (M) Nile Red staining of the control and MZglut2 fish at 3 mpf. White arrows: visceral adipose tissue. (N) The bar chart represents the relative fluorescence intensity of the control and MZglut2 fish after Nile Red staining (n = 8). (O) Liver triglyceride content at 3 mpf (n = 6). (P) Oil Red O staining of liver sections for distribution of lipid droplets in the control and MZglut2 fish at 3 mpf. (Q) The bar chart represents the relative lipid droplet areas of control and MZglut2 fish after Oil Red O staining (n = 8). A two-tailed unpaired t-test was used to detect significance. ****P < 0.0001.***P < 0.001. **P < 0.01. *P < 0.05. ns, no significance.

glut2-deletion results in enhanced AMPK-mediated catabolic metabolism in MZglut2 fish. (A) The quantification of amino acid metabolites is based on the observations of metabolomics in the liver of the control and MZglut2 fish at 3 mpf (n = 6). (B–D) The level of P-AMPK and β-ACTIN proteins in the liver and muscle of the control and MZglut2 fish (n = 3). (E) The ATP content in the liver of control and MZglut2 fish (n = 6). (F) The ATP content in the muscle of the control and MZglut2 fish (n = 6). (G) The transcriptional expression levels of mtor, bcat1, bcat2, bckdha, bckdk, glud1b, asns, atf4a, eif4ebp3, and murf1a in muscle of control and MZglut2 fish at 3 mpf (n = 6). (H) The measurement of the crude protein content of the control (n = 9) and MZglut2 (n = 8) fish at 3 mpf. (I) Representative images of H&E staining of the control and MZglut2 fish at 3 mpf. (J) The bar chart represents the total area of muscle mass in the body cross-section (n = 6). A two-tailed unpaired t-test was used to detect significance. ****P < 0.0001.***P < 0.001. **P < 0.01. *P < 0.05. ns, no significance.

glut2-deletion resulted in slower movement activity but increased oxygen consumption in MZglut2 fish. (A, B) Pathway monitoring of the control and MZglut2 fish continued for 5 min at 3 mpf. (C, D) The distance covered with slow-mild speed or large speed of the control fish (n = 9) and MZglut2 (n = 8). Slow-mild speed (0–2 cm/s) and large speed (> 2 cm/s) of the control and MZglut2 fish. (E, F) The oxygen consumption rate of basic level and 6 hpp in the control and MZglut2 fish at 3 mpf (n = 4). A two-tailed unpaired t-test was used to detect significance. **P < 0.01. *P < 0.05.

A hypothetical diagram showing how glut2-deletion induces remodeling of nutrient metabolism in zebrafish (the blue arrow means downregulation and the red arrow means upregulation).