Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production, and regulation of blood sugar control. In addition, ZAK^-/- male mice present a lean phenotype. Our work highlights impaired ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation. Summary Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. To achieve energy homeostasis in the face of such challenges, a large degree of metabolic flexibility is required. 1 , 2 This flexibility necessitates fine-tuning by cellular sensors of energy and nutrient availability that mediate switches between anabolism and catabolism. Among these, the mTOR kinase monitors the availability of amino acids and directly impacts protein translation and other anabolic reactions. 3 Conversely, the AMPK kinase responds to low energy levels by increasing glucose uptake and lipid oxidation. 4 In mammalian organisms, the liver is the main site of glucose storage and release, while adipose tissues store excess energy in the form of lipids. Results The RSR is activated by starvation-induced ribosome stalling Incubation of human U2OS and HeLa cells in an Earle’s balanced salt solution (EBSS) starvation medium for several hours led to a marked activation of p38 and JNK, and these effects were abolished in corresponding ZAK knockout (KO) cells ( Figures 1 A and S1 A). This response appeared to be related to amino acids availability, as incubation with histidinol or medium depleted of either glutamine, leucine or lysine, arginine, and leucine in combination (÷AA) all decreased global translation and triggered RSR signaling ( Figures 1 B, 1C, and S1 A–S1C), as also previously reported for glutamine starvation. Amino acid starvation has previously been reported to be associated with both stalling and collision of ribosomes. 9 , 14 These structures can be distinguished by in vitro digestion of polysomes with micrococcal nuclease (MNase), which leaves the mRNA spanning disomes and trisomes intact. 7 Using this method, we did not observe any signs of collided ribosomes in cells incubated in ÷AA medium, even in the absence of the disassembly factor ASCC3 15 ( Figures 1 F and S1 H). However, inhibition of the ISR kinase GCN2, allowing new cap-dependent initiation of translation, was accompanied by ribosome collision, and this effect was exacerbated by ASCC3 knockdown ( Figure 1 F). These results indicate that ribosome stalling is also the major translational aberration induced by EBSS. However, conversion of these stalls to collisions was accompanied by elevated ZAK-dependent p38 activation ( Figure 1 H). We propose that both full starvation and amino acid starvation induce ribosome stalling that activates the RSR. While not an absolute requirement, collided ribosomes appear to provide a more robust platform for ZAKα activation compared with stalled ribosomes ( Figure 1 I). EBSS and amino acid starvation-induced activation of the energy-sensing kinase AMPK also required ribosome binding and kinase activity of ZAKα ( Figures 2 C, S2 G, and S2H). Downstream of ZAKα, control over AMPK appeared to be exerted by JNK kinases, as application of a JNK inhibitor, but not p38 inhibitor, phenocopied the effect of ZAK deletion ( Figure 2 D). Inhibition of mTOR catalytic activity by torin caused spontaneous activation of RSR signaling, and torin-induced AMPK activation was dependent on ZAK and JNK activity ( Figures 2 E and 2F). Of note, treatment of cells with torin did not result in ribosome collision, even in the presence of GCN2 inhibitor ( Figure S2 I). 31 Our analysis indicated lower footprint coverage at the 5′ end of the coding sequence in leucine-starved mice ( Figure 3 I), consistent with ISR activation reducing overall translation initiation. To probe for ribosomal decoding defects associated with specific codons or amino acids, we first assessed whether the predicted A-sites of footprints showed diet-dependent changes in their codon distributions. Overall, codons were very similarly occupied in full diet and leucine-starved animals ( Figure 3 J). Of note, the actually depleted amino acid, leucine, was not affected ( Figures 3 K and 3L), reminiscent of previous findings from leucine-deprived cell cultures. All scale bars, 50 μm. Data are plotted as mean and all error bars represent the SEM. ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p > 0.001, ∗∗∗∗ p < 0.0001 in two-way ANOVA. See also Figure S4 . Discussion Our results point to a role for ZAKα-activated JNK in potentiating AMPK activation upon mTOR inactivation. While mTOR itself responds directly to amino acid availability, ZAKα activation appears to rely on