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The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1-4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca +2 concentration ([Ca +2 ]i) mediated by G q11 coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombininduced [Ca +2 ]i increase and demonstrated the calcium-dependent activation of -calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of -spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel -calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.

Hepatic steatosis is characterized by triglyceride accumulation within hepatocytes in response to a high calorie intake, and it may be related to intestinal microbiota disturbances. The prebiotic inulin is a naturally occurring polysaccharide with a high dietary fiber content. Here, we evaluate the effect of inulin on the intestinal microbiota in a non-alcoholic fatty liver disease model. Mice exposed to a standard rodent diet or a fat-enriched diet, were supplemented or not, with inulin. Liver histology was evaluated with oil red O and H&E staining and the intestinal microbiota was determined in mice fecal samples by 16S rRNA sequencing. Inulin treatment effectively prevents liver steatosis in the fat-enriched diet group. We also observed that inulin re-shaped the intestinal microbiota at the phylum level, were Verrucomicrobia genus significantly increased in the fat-diet group; specifically, we observed that Akkermansia muciniphila increased by 5-fold with inulin supplementation. The family Prevotellaceae was also significantly increased in the fat-diet group. Overall, we propose that inulin supplementation in liver steatosis-affected animals, promotes a remodeling in the intestinal microbiota composition, which might regulate lipid metabolism, thus contributing to tackling liver steatosis.

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