Leptin receptor-deficient (knockout) zebrafish: Effects on nutrient acquisition

Abstract

In mammals, knockout of LEPR results in a hyperphagic, morbid obese, and diabetic phenotype, which supports that leptin plays an important role in the control of appetite and energy metabolism, and that its receptor, LEPR, mediates these effects. To date, little is known about the role(s) of lepr in teleost physiology. We investigated a zebrafish (Danio rerio) homozygous lepr knockout (lepr−/−) line generated by CRISPR/Cas9 in comparison to its wt counterpart with respect to nutrient acquisition, energy allocation, and metabolism. The metabolic characterization included oxygen consumption rate and morphometric parameters (yolk sac area, standard length, wet weight, and condition factor) as proxies for use and allocation of energy in developing (embryos, larvae, and juveniles) zebrafish and showed no particular differences between the two lines, in agreement with previous studies. One exception was found in oxygen consumption at 72 hpf, when zebrafish switch from embryonic to early larval stages and food-seeking behavior could be observed. In this case, the metabolic rate was significantly lower in lepr−/− than in wt. Both phenotypes showed similar responses, with respect to metabolic rate, to acute alterations (22 and 34 °C) in water temperature (measured in terms of Q10 and activation energy) compared to the standard (28 °C) rearing conditions. To assess lepr involvement in signaling the processing and handling of incoming nutrients when an exogenous meal is digested and absorbed, we conducted an in vivo analysis in lepr−/− and wt early (8 days post-fertilization) zebrafish larvae. The larvae were administered a bolus of protein hydrolysate (0%, 1%, 5%, and 15% lactalbumin) directly into the digestive tract lumen, and changes in the mRNA expression profile before and after (1 and 3 h) administration were quantified. The analysis showed transcriptional differences in the expressions of genes involved in the control of appetite and energy metabolism (cart, npy, agrp, and mc4r), sensing (casr, t1r1, t1r3, t1r2-1, t1r2-2, pept1a, and pept1b), and digestion (cck, pyy, try, ct, and amy), with more pronounced effects observed in the orexigenic than in the anorexigenic pathways, suggesting a role of lepr in their regulations. Differences in the mRNA levels of these genes in lepr−/− vs. wt larvae were also observed. Altogether, our analyses suggest an influence of lepr on physiological processes involved in nutrient acquisition, mainly control of food intake and digestion, during early development, whereas metabolism, energy allocation, and growth seem to be only slightly influenced.