DUAL ACTIVATION OF GCGR/GLP1R SIGNALING AMELIORATES INTESTINAL FIBROSIS VIA METABOLIC REGULATION OF HISTONE H3K9 LACTYLATION IN EPITHELIAL CELLS

BACKGROUND
Intestinal fibrosis is a significant clinical challenge in inflammatory bowel diseases, but no effective anti-fibrotic therapy is currently available. GCGR and GLP1R are both peptide hormone receptors involved in energy metabolism of epithelial cells. However, their role in intestinal fibrosis and the underlying mechanisms remain largely unexplored.

METHODS
Intestinal tissues from patients with Crohn's disease (CD) (n=20) were obtained. AAV9-Glp1r-shRNA, AAV9-Gcgr-shRNA or AAV9-NC-shRNA were used to transfect mice. Murine colons were analyzed by single-cell RNA sequencing (seq) and metabonomics. Glycolytic metabolism of epithelial cells was analyzed with bulk RNAseq and seahorse-XF technology, while lactylation levels were verified using antibodies against diverse forms of lactylated histones. Then a range of GCGR/GLP1R dual-target ultralong-acting peptides were synthesized, and their agonistic activities were assessed in HEK293 cells that stably expressed either human GLP1R or GCGR, using a cAMP response element-driven luciferase reporter. The antifibrotic activity of the leading peptide 1907B was functionally evaluated in vitro and in vivo.

RESULTS
GCGR and GLP1R were reduced in the stenotic ileum of patients with CD as well as in the fibrotic colon of mice with chronic colitis. In the chronic model, Gcgr/Glp1r dual knockdown worsened the degree of fibrosis in mice, and increased glycolysis-related metabolites, with lactate exhibiting the most prominent alterations. In the TGFβ1-induced epithelial cell model, exogenous and endogenous lactate induced fibrotic genes expression and overwhelming histone lactylation in H3K9 (H3K9la). Further, cells overexpressing H3.1 (K9R) exhibited lower levels of fibrotic genes and proteins after treatment with lactate compared with the H3.1 WT group. Then 1907B, the leading GCGR/GLP1R dual-target peptide, normalized the ATP production rate of glycolysis, extracellular lactate, H3K9la levels of promoters related to fibrosis genes, and further ameliorated intestinal fibrosis in vivo.

CONCLUSION
We uncovered the role of GCGR/GLP1R in regulating EMT involved in intestinal fibrosis via histone lactylation. Simultaneously activating GCGR/GLP1R with the novel dual agonist 1907B holds promise as a treatment strategy for alleviating intestinal fibrosis.