FIGURE SUMMARY
Title

Leptin regulates glucose homeostasis via the canonical Wnt pathway in the zebrafish

Authors
Kamstra, K., Rizwan, M.Z., Grattan, D.R., Horsfield, J.A., Tups, A.
Source
Full text @ FASEB J.

Leptin treatment ameliorates hyperglycemia in male and female zebrafish. (A, B) Blood glucose values of male (right) and female (left) wild-type zebrafish over time following immersion in a 1% glucose solution. On the third day, 1 h before blood sampling, fish were injected intraperitoneally with recombinant mouse leptin (2 mg/kg) or vehicle. *< .05, repeated-measures one-way ANOVA. Data are displayed as mean ± SEM. (C, D) Male (right) and female (left) lepanz301 fish. (E, F) Male (right) and female (left) lepbnz302 fish. (G, H) Male (right) and female (left) leprnz303 fish

Regulation of glucose homeostasis and body weight in adult zebrafish. (A) Glucose tolerance in normal-fed lepanz301 fish, lepbnz302 fish, leprnz303 fish, and wild-type controls (n = 12). (B) Baseline blood glucose levels of normal-fed and overfed lepanz301 fish, lepbnz302 fish, leprnz303 fish, and wild-type controls. ****< .0001, two-way ANOVA. (C) Glucose tolerance in overfed lepanz301 fish, lepbnz302 fish, leprnz303 fish, and wild-type controls (n = 12). (D) Area under the curve of (A) and (C). *< .05, one-way ANOVA; #< .05, two-way ANOVA. (E) Body weights of lepanz301 fish, lepbnz302 fish, leprnz303 fish, and wild-type control on a 6-week normal diet or overfeeding regime. ***< .001, repeated-measures ANOVA. (F) Standard length of normal-fed and overfed lepanz301 fish, lepbnz302 fish, leprnz303 fish, and wild-type controls at 0, 3, and 6 weeks. *< .05, one-way ANOVA. (G–J) Examples of leptin receptor-deficient and wild-type zebrafish at the start and end of the overfeeding regime

Wnt pathway activation by leptin in Tg(7xTCFXla. Siam:nlsmCherry)ia5 larvae. (A) 5 dpf Tg(7xTCFXla. Siam:nlsm Cherry)ia5 larvae treated with vehicle (Cortland salt solution). Left: bright field image, with anatomical landmarks encircled; Middle: Epifluorescence image, with white arrow indicating teeth protrusion; Right: Magnification of yellow box in middle image, with hypothalamus and heart encircled. Scale bar = 500 μM. (B) 5 dpf Tg(7xTCFXla. Siam:nlsm Cherry)ia5 larvae treated with 100 nM recombinant leptin for 2 h. (C) 5 dpf CRISPR-mediated leptin receptor-deficient Tg(7xTCFXla. Siam:nlsm Cherry)ia5 larvae treated with 100 nM recombinant leptin for 2 h. (D) 5 dpf CRISPR-mediated leptin receptor-deficient Tg(7xTCFXla. Siam:nlsm Cherry)ia5 larvae treated with 10 μM LiCl for 2 h. (E) Fluorescence intensity in the hypothalamus of differentially treated 5 dpf Tg(7xTCF-Xla. Siam:nlsmCherry)ia5 larvae. A–B = < .05, one-way ANOVA. (F) Fluorescence intensity in the heart of differentially treated 5 dpf Tg(7xTCF-Xla. Siam:nlsmCherry)ia5 larvae. A–B = < .05, one-way ANOVA

Activation of canonical Wnt signaling improves glucose tolerance in lepanz301 fish, lepbnz302 fish, and leprnz303 zebrafish. (A) Glucose tolerance of adult male lepanz301 zebrafish (n = 6). Fish were treated 10 μM LiCl (3 h before glucose injection), 0.6 g/L of recombinant mouse leptin dissolved in Cortland salt solution (1 h before glucose injection), vehicle only, or a combination of LiCl and leptin. Following 0.5 mg/g glucose injection, blood samples were taken at 30, 90, and 180 min post-injection. (B) Same as (A), but for lepbnz302 fish. (C) Same as (A) and (B), but for leprnz303 fish. (D) Area under the curve of (A). **< .001, one-way ANOVA. (E) Area under the curve of (B). *< .05, one-way ANOVA. (F) Area under the curve of (C). **< .001, one-way ANOVA. (G) Blood glucose values of adult male lepanz301 zebrafish (n = 6) over the course of a 4-day immersion in a 1% glucose solution. On the third day, fish were exposed to 10 μM LiCl for three h before daily blood sampling. One hour before blood sampling, fish were injected intraperitoneally with 0.6 g/L of recombinant mouse leptin dissolved in Cortland salt solution, or with vehicle only. *< .05, repeated-measures ANOVA. (H) Same as (G), but for lepbnz302 fish. *< .05, repeated-measures ANOVA. (I) Same as (G) and (H), but for leprnz303 fish. *< .05, repeated-measures ANOVA. (J) Blood glucose levels of (G), comparing pre-treatment (day 2) and post-treament (day 3). (K) Same as (J) but relating to (H). (L) Same as (J) and (K) but relating to (I)

Glucose tolerance in wild-type zebrafish following Wnt pathway manipulation and leptin treatment. (A) Glucose tolerance of adult wild-type male zebrafish (n = 6). Fish were treated 10 μM LiCl (3 h before glucose injection), 0.6 g/L of recombinant mouse leptin dissolved in Cortland salt solution (1 h before glucose injection), vehicle only, or a combination of LiCl and leptin. Following 0.5 mg/g glucose injection, blood samples were taken at 30, 90, and 180 min post-injection. (B) Area under the curve of (A). A–B = < .05, one-way ANOVA. (C) Blood glucose values of adult wild-type male zebrafish (n = 6) over the course of a 4-day immersion in a 1% glucose solution. On the third day, fish were exposed to 10 μM LiCl for 3 h before daily blood sampling. One hour before blood sampling, fish were injected intraperitoneally with 0.6 r/L of recombinant mouse leptin dissolved in Cortland salt solution, or with vehicle only. (D) Glucose tolerance of adult wild-type male zebrafish (n = 6). Fish were treated 10 μM PNU74654 (3 h before glucose injection), 0.6 g/L of recombinant mouse leptin dissolved in Cortland salt solution (1 h before glucose injection), vehicle only, or a combination of PNU74654 and leptin. (E) Area under the curve of (D). A–B and A–C = < .05, one-way ANOVA. (F) Blood glucose values of adult wild-type male zebrafish (n = 6) over the course of a 4-day immersion in a 1% glucose solution with exposure to 10 μM PNU74654. (G) Glucose tolerance of adult wild-type male zebrafish (n = 6). Fish were treated with 10 μM pyrvinium pamoate (3 h before glucose injection), 0.6 g/L of recombinant mouse leptin dissolved in Cortland salt solution (1 h before glucose injection), vehicle only, or a combination of pyrvinium pamoate and leptin. (H) Area under the curve of (G). A–B and A–C = < .05, one-way ANOVA. (I) Blood glucose values of adult wild-type male zebrafish (n = 6) over the course of a 4-day immersion in a 1% glucose solution with exposure to 10 μM pyrvinium pamoate. (J) Comparison of blood glucose levels in (C), (F), and (I), pre-treatment (day 2), and post-treatment (day 3). *< .05, repeated-measures ANOVA

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ FASEB J.