The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish

Abstract

Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel-Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells.

Fig. 1.

Characterization of zebrafish lkb1 mutations. (A) Whole-mount in situ hybridizations show decreased lkb1 mRNA expression in 3-dpf lkb1-mutant larvae. (B) Western blot analysis of AMPK and ACC upon KCN treatment. KCN treatment results in activation of the AMPK pathway in 3-dpf WT larvae but not in 3-dpf lkb1-mutant larvae.

Fig. 2.

The intestinal architecture of lkb1 mutants resembles that of starved WT larvae. (A, C, and E) High-power images depicting the liver and intestine of live larvae of the indicated genotypes; anterior is to the left. Red brackets demarcate the thickness of the intestinal wall, and livers are outlined. At 7 dpf, lkb1-mutant larvae (C) exhibit a small, dark liver (L) and flattened intestine (asterisk), as do starved 11-dpf WT larvae (E). (B, D, and F) H&E staining of sagittal sections of the intestine. Note loss of intestinal folding in 7-dpf lkb1- mutant larvae and in starved WT larvae. Sb, swim bladder.

Fig. 3.

lkb1 mutants exhibit hallmarks of response to starvation and high metabolic rate. (A) Transverse vibratome sections of PAS-stained WT and lkb1-mutant livers at 5 and 7 dpf. At 7 dpf the WT liver still contains a moderate amount of glycogen, but glycogen is depleted in the lkb1-mutant liver. (B) Transverse vibratome sections of ORO-stained WT and lkb1-mutant livers on days indicated. Note strong lipid accumulation in the lkb1-mutnt liver at 7 dpf and in starved WT liver at 11 dpf. (C) Lkb1 mutants exhibit high metabolic rate. Histograms depict acid production rates of WT and lkb1-mutant larvae at different days of development. The rate of acid production correlates with metabolic rate and was calculated as described in SI Materials and Methods.

Fig. 4.

The zebrafish TOR (zTOR) pathway activity in lkb1 mutants resembles that in starved WT larvae. (A) Western blot analysis with antibodies against phospho-AMPK and phospho-ACC of total protein lysates from embryos at the indicated days and genotypes. AMPK and ACC are not phosphorylated in WT or lkb1-mutant larvae at 5 or 7 dpf. Prolonged fasting induces phosphorylation of AMPK and ACC in 11-dpf WT larvae. Elevated expression of AMPK in 7-dpf lkb1-mutant larvae is detected. (B) Western blot analysis using anti–phospho-p70S6K, anti-p70S6K, anti–phospho-4EBP1, anti-4EBP1, anti–phospho-RS6, and anti-RS6 antibodies of total protein lysates from embryos at the indicated days and genotypes. (C) Sections of livers obtained from WT and lkb1-mutant embryos at the indicated days stained with anti–phospho-RS6 and anti–phospho-4EBP1.

Fig. 5.

Deregulation of PI3K signaling in lkb1 mutants. (A) Transverse sections of WT and lkb1-mutant livers at indicated days of development stained with an antibody against phospho-AKT. Strong phospho-AKT staining is detected in WT and lkb1-mutant livers at 5 dpf. WT liver is strongly stained at 7 dpf, whereas phospho-AKT staining is barely detectable in 7-dpf lkb1-mutant liver and in starved WT at 11 dpf. (B) Inhibition of PI3K signaling leads to a starvation-like phenotype in WT larvae. WT larvae at 7 dpf treated for 3 d with either LY29 or its inactive analog LY30. LY29 treatment of WT larvae at 4 dpf leads to dark liver (arrowheads) and abnormal hepatic steatosis as revealed by ORO staining. Treatment with the inactive analog LY30 has no effect in the morphology of the larvae.

Fig. 6.

Loss of vhl suppresses manifestation of the lkb1 phenotype. (A) High-power images depicting the liver and intestine of live larvae of the indicated genotypes at 7 dpf; anterior is to the left. Note that some yolk (Y) is still present. The intestine is folded, and the liver is clear in the lkb1/vhl larva. (B) Graph showing ATP levels as measured in relative light units (RLU). The lkb1 embryos have very low ATP levels; ATP levels are decreased in vhl and lkb1/vhl embryos but are slightly higher than in lkb1-mutant embryos. (C) Graph representing the metabolic rate of larvae of different genotypes at 3 dpf. The lkb1-mutant larvae have an increased metabolic rate as compared with WT larvae. The vhl larvae display a dramatically low metabolic rate, and the lkb1/vhl larvae also display a low metabolic rate.