F Thomas Wunderlich - C 19

The obesity-induced leaky gut is the source of the low-grade metaflammation that impairs systemic insulin sensitivity and is responsible for numerous obesity-associated complications.

Therefore, tightening of the intestinal epithelium in obesity might be a promising therapeutic approach to prevent such severe consequences. Thus, understanding the crosstalk between regenerative and metabolic regulation of intestinal integrity is essential to interfere with candidate gene expression to ultimately prevent gut leakage in obesity.

The intestinal epithelial cell layer is constantly regenerating from intestinal stem cells (ISC) and protects the organism against invading and commensal bacteria. Interestingly, obesity is accompanied by a leaky gut and a reasonable hypothesis even states that the obesity-induced low-grade metabolic inflammation (metaflammation) is derived from gut derived bacteria.

We have demonstrated previously that intestinal insulin resistance promotes colorectal cancer by impairing intestinal integrity. Our report suggests a crosstalk between the cell-cell contact sensitive regenerative Hippo Yap/Taz signaling pathway and metabolic Insulin receptor/FoxO1 mediated transcriptional regulation of genes in this process.

We propose to use a sophisticated dual recombinase approach in lineage tracing experiments and to dissect the specific gene expression profile regulated by Yap and FoxO1 under normal and insulin resistant conditions. To this end, we will inducibly express EGFP-RPL10a in Yap-expressing intestinal cells in the presence or absence of insulin resistance, allowing for dissecting the stage and cell type-specific gene expression profile that is able to regenerate the intestinal epithelium. The functional consequences of candidate genes identified by this screen will be revealed in metabolic mouse experiments.

1. Examine intestinal regenerative capacity under normal, diet-induced, and genetic insulin resistance using lineage tracing technology.
   - We will genetically label intestinal stem cells and verify their regenerative capacity under diet-induced and genetic insulin resistance via examination of intestinal cell types such as enterocytes, stem-, enteroendocrine- goblet- and Paneth-cells.
2. dentify differential gene expression dependent on simultaneous nuclear Yap and nuclear excluded FoxO1 transcriptional regulation to identify candidate genes that impact on gut barrier function in insulin resistant states.
   - For, we will examine Yap/FoxO1 regulated gene expression in intestinal stem cells via pull down of actively translating ribosomes using intestinal translational ribosome affinity purification technology in diet-induced and genetic insulin resistance.  
3. Verify identified candidate genes in vivo and proof their specific role on intestinal barrier function.

Obesity is a steadily increasing health burden of western societies that predisposes to numerous disorders. The obesity-induced low-grade metabolic inflammation (metaflammation) has been shown to impair systemic insulin action and is causative for the development of insulin resistance and other fatal complications. One hypothesis states that in obesity, the intestinal epithelial cell (IEC) layer becomes leaky, thereby exposing the mucosal immune system to the commensal microbiota to initiate metaflammation. The intestinal inflammation drains into the liver via the portal vein from where it spreads throughout the body. The intestine is a highly regenerative organ where the loss of cell-cell contacts shuts down the Hippo pathway to initiate a transcriptional program via Yap/Taz in intestinal stem cells (ISC) to restore epithelial integrity.

ISCs reside at the bottom of crypts and are able to regenerate every IEC type such as enterocytes, goblet cells, neuroendocrine precursors or paneth cells upon demand. Recent research has highlighted the importance of the Hippo pathway in ISCs in the regulation of intestinal regeneration (Gregorieff et al., 2015).  In the presence of cell-cell contact, the Hippo pathway is activated and constantly degrades the transcriptional coactivators Yap/Taz, whereas intestinal loss of cell-cell contact i.e. via cell death deactivates the Hippo pathway to stabilize Yap/Taz leading to regenerative gene expression such as Sca1/Ly6a to maintain epithelial integrity (Yui et al., 2018). Strikingly, a so far unidentified EGF-R driven signaling node in ISCs is needed in parallel to synergistically promote intestinal regeneration (Gregorieff et al., 2015).

The EGF-R is a receptor tyrosine kinase (RTK) that exhibits similar signaling capacities as the RTKs insulin receptor (IR) and Igf1R including AKT-mediated phosphorylation and subsequent nuclear exclusion of FoxO1. However, double deficiency of EGF-R and Igf1R in IECs failed to affect intestinal regeneration indicating that insulin signaling may compensate to regenerate the intestines from these mice (Sun et al., 2015). Surprisingly, intestinal epithelial cell (IEC) specific inactivation of IR drives a prolonged regenerative YAP-mediated program in ISCs upon challenge that increases colorectal cancer burden (Yassin et al., 2018). Our own results suggest the hypothesis how synergistic Hippo and RTK signaling might drive regenerative and metabolic programming of ISCs.

We show that genetic and diet-induced insulin resistance retains FoxO1 in the nucleus and that nuclear FoxO1 via expression of an insulin insensitive FoxO1ADA recapitulates the exacerbated colorectal cancer phenotype in our mouse models (Ostermann et al., 2019). Collectively, these results strongly suggest that simultaneous Hippo off (nuclear Yap) and RTK on (FoxO1 nuclear exclusion) in ISCs is required for controlled transcriptional regulation of intestinal regenerative capacity that might be impaired in obesity/insulin resistance to account for the obesity-induced leaky gut. Identification of candidate genes regulated by this synergistic transcriptional regulation might provide novel therapeutic strategies to prevent obesity-induced leaky gut, metaflammation and such severe complications as the development of insulin resistance and Type 2 diabetes mellitus.

1. Gregorieff, A., Liu, Y., Inanlou, M.R., Khomchuk, Y., and Wrana, J.L. (2015). Yap-dependent reprogramming of Lgr5(+) stem cells drives intestinal regeneration and cancer. Nature 526, 715-718.
2. Ostermann AL, Wunderlich CM, Schneiders L, Vogt MC, Woeste MA, Belgardt BF, Niessen CM, Martiniy B, Schauss AC, Frommolt P, Nikolaev A, Hövelmeyer N, Sears RC, Koch PJ, Günzel D, Brüning JC, Wunderlich FT (2019) Intestinal insulin/IGF1 signalling through FoxO1 regulates epithelial integrity and susceptibility to colon cancer. Nature Metabolism
3. Sun, R.C., Diaz-Miron, J.L., Choi, P.M., Sommovilla, J., Guo, J., Erwin, C.R., and Warner, B.W. (2015). Both epidermal growth factor and insulin-like growth factor receptors are dispensable for structural intestinal adaptation. J Pediatr Surg 50, 943-947.
4. Yassin, M., Sadowska, Z., Tritsaris, K., Kissow, H., Hansen, C.H.F., Forman, J.L., Rogler, G., Troelsen, J.T., Pedersen, A.E., and Olsen, J. (2018). Rectal insulin instillation inhibits inflammation and tumor development in chemically-induced colitis. J Crohns Colitis.
5. Yui, S., Azzolin, L., Maimets, M., Pedersen, M.T., Fordham, R.P., Hansen, S.L., Larsen, H.L., Guiu, J., Alves, M.R.P., Rundsten, C.F., et al. (2018). YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell Stem Cell 22, 35-49 e37.