Autophagy compensates for Lkb1 loss to maintain adult mice homeostasis and survival

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11) is the major energy sensor for cells to respond to metabolic stress. Autophagy degrades and recycles proteins, macromolecules, and organelles for cells to survive starvation. To assess the role and cross-talk between autophagy and Lkb1 in normal tissue homeostasis, we generated genetically engineered mouse models where we can conditionally delete Stk11 and autophagy essential gene, Atg7, respectively or simultaneously, throughout the adult mice. We found that Lkb1 was essential for the survival of adult mice, and autophagy activation could temporarily compensate for the acute loss of Lkb1 and extend mouse life span. We further found that acute deletion of Lkb1 in adult mice led to impaired intestinal barrier function, hypoglycemia, and abnormal serum metabolism, which was partly rescued by the Lkb1 loss-induced autophagy upregulation via inhibiting p53 induction. Taken together, we demonstrated that autophagy and Lkb1 work synergistically to maintain adult mouse homeostasis and survival.

vessels and death at E12.5, which is attributed to the reduced Tgfb signaling in yolk sac (1, 6).

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Liver-specific deficiency of Lkb1 causes impaired glucose metabolism (7). Muscle-specific 11 deletion of Lkb1 results in lower fasting blood glucose and insulin levels, along with increased 12 glucose uptake through muscles (8). Lkb1 loss in hematopoietic stem cells causes dysfunctional 13 mitochondria, leading to pancytopenia due to reduced levels of ATP, fatty acids and nucleotides 14 (3,9,10). Taken together, tissue-specific knockout studies underscore the importance of Lkb1 in 15 tissue homeostasis, metabolism, and stem cell maintenance. Somatic LKB1 mutations are related 16 with a number of human cancers; however, tissue-specific removal of Lkb1 in mice does not 17 necessarily lead to tumor formation (11).

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Given that both Lkb1 signaling and autophagy play indispensable roles in maintaining tissue 25 energy homeostasis, we began to investigate the interaction of Lkb1 signaling and autophagy in 26 4 supporting homeostasis of adult mice. We engineered mice to conditionally (Tamoxifen (TAM)-1 inducible) and systemically delete Lkb1 and Atg7, either respectively or simultaneously. Same as 2 previous report (16), systemic Atg7 ablation led to extensive liver and muscle damage, and 3 neurodegeneration starting at 6 weeks post-deletion, and limited mouse survival to 3 months.

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Given that AMPK is required for autophagy activation, Lkb1 deficiency leads to the loss of AMPK

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At 15 days post-TAM administration, compared with WT control mice, there was a significant loss 15 of body weight in Lkb1 -/mice, which was further deteriorated by the loss of Atg7 (Fig. 2b).

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Acute autophagy ablation aggravates Lkb1-deficiency-induced loss of secretory cell 22 structure in small intestine

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To elucidate the underlying mechanism by which Atg7 and Lkb1 ablation alone or in concurrent 24 impacts the mouse survival, we examined the histology of different tissues. After short-period (15 25 days) of protein deletion, which is before the death of Atg7 -/-;Lkb1 -/mice, the damage of tissues 7 was not observed in Atg7-deficient mice ( Supplementary Fig. 2a). Moreover, except for the 1 intestine ( Fig. 3a), most of the tissues in both Lkb1 -/and Atg7 -/-;Lkb1 -/mice were not visibly

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The structure of the small intestine was extremely damaged by Lkb1 deletion alone or co-24 deletions of Lkb1 and Atg7 ( Fig. 3a-d), which could be due to less regeneration from intestinal 25 stem cells or increased cell death. Cell death in small intestine occurs through apoptosis at the tip of the villi which eventually leads to shedding of dead cells into the lumen (29, 30). We found 1 that there was a significant increase of apoptotic cell death in the tip of the villi in Atg7 -/-;Lkb1 -/-2 mice compared with Lkb1 -/mice (Fig. 3e). Intestinal crypt is the region for cell division and 3 migration to upper sites of the villi (31). Compared with the WT control mice, we did not observe 4 any significant difference in the cell proliferation rate in the crypt of the mice lacking either Atg7 5 or Lkb1 alone or in combination ( Supplementary Fig. 2d).

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Taken together, we show that Lkb1 is necessary for maintaining the structural integrity of the 8 intestine and that autophagy activation partly compensates for the severe intestinal phenotype 9 induced by the loss of Lkb1.  we performed serum metabolomics in fasting state after short-term deletion of the genes. Of the 10 90 metabolites we examined, we found that acute deletion of Atg7 or Lkb1 alone significantly 11 decreased the levels of most essential and non-essential amino acids and some metabolites 12 involved in urea cycle and glycolysis ( Fig. 5a-d). Interestingly, we found that the reduced levels In this study, we demonstrated the intermingled essential and systemic roles of Lkb1 and 5 autophagy in the maintenance of mouse homeostasis and survival via conditional whole-body 6 deletion of Lkb1 and Atg7 in adult mice (Fig. 6). We found that acute Lkb1 loss led to damaged 7 intestinal epithelium barrier and increased infection, and alteration in metabolic pathways 8 necessary for maintaining host homeostasis, which was partially rescued by autophagy activation 9 via inhibiting p53 induction. Thus, autophagy upregulation compensates for the acute Lkb1 loss 10 to temporarily support the survival of adult mice.

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An accumulating body of evidence suggests that Lkb1 phosphorylates AMPK and activates 13 autophagy in response to energy crises (1-3, 18, 21). However, in addition to Lkb1, CaMKK, and 14 TAK1 also trigger AMPK activation (22). Here we observed an upregulation of autophagy in Lkb1-15 deficient mice, suggesting that Lkb1-AMPK-mTORC1-autophagy axis may not be the only or the 16 critical effector of Lkb1-mediated maintenance of adult mice homeostasis in our setting. This is 17 consistent with a previous study that Lkb1-mediated energy metabolism is largely independent of 18 Lkb1 regulation of AMPK and mTORC1 signaling in mouse hematopoietic stem cells (9).

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Hematopoietic stem cell-specific deletion of Lkb1 led to limited mouse survival for up to 28 days due to pancytopenia (9). However, in our study, although mice died within 4 weeks after Lkb1 1 ablation, no obvious reduction in different types of blood cells was observed. Instead, serum 2 metabolomics analysis showed significant reduction in most of the essential and non-essential

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Histology and immunohistochemistry

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Paraffin-embedded tissue sections were prepared as described previously (12)   compare the progression-free survival was calculated using the log-rank test. The mass spectra 23 were analyzed by MAVEN software and the peak area of each detected metabolite was obtained.

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Statistical significance of metabolites was determined by a paired two-tailed Student's t-test, and 25 five mice from each genotype were used. P-value of <0.05 was considered statistically significant.       Representative IHC of p53 with lower magnification in brain, heart, intestine, kidney, liver, muscle and spleen of WT control, Atg7 -/-, Lkb1 -/-, and Atg7 -/-;Lkb1 -/adult mice shows the increase of nuclear p53 in Atg7-ablated tissues.