PAR2 promotes impaired glucose uptake and insulin resistance in NAFLD through GLUT2 and Akt interference.

BACKGROUND AND AIMS: Insulin resistance and poor glycemic control are key drivers of the development of NAFLD and have recently been shown to be associated with fibrosis progression in NASH. However, the underlying mechanisms involving dysfunctional glucose metabolism and relationship with NAFLD/NASH progression remain poorly understood. We set out to determine whether protease-activated receptor 2 (PAR2), a sensor of extracellular inflammatory and coagulation proteases, links NAFLD and NASH with liver glucose metabolism. APPROACH AND RESULTS: Here, we demonstrate that hepatic expression of PAR2 increases in patients and mice with diabetes and NAFLD/NASH. Mechanistic studies using whole-body and liver-specific PAR2-knockout mice reveal that hepatic PAR2 plays an unexpected role in suppressing glucose internalization, glycogen storage, and insulin signaling through a bifurcating Gq -dependent mechanism. PAR2 activation downregulates the major glucose transporter of liver, GLUT2, through Gq -MAPK-FoxA3 and inhibits insulin-Akt signaling through Gq -calcium-CaMKK2 pathways. Therapeutic dosing with a liver-homing pepducin, PZ-235, blocked PAR2-Gq signaling and afforded significant improvements in glycemic indices and HbA1c levels in severely diabetic mice. CONCLUSIONS: This work provides evidence that PAR2 is a major regulator of liver glucose homeostasis and a potential target for the treatment of diabetes and NASH.

Targeting Liver Fibrosis with a Cell-penetrating Protease-activated Receptor-2 (PAR2) Pepducin.

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

PAR2 controls cholesterol homeostasis and lipid metabolism in nonalcoholic fatty liver disease.

OBJECTIVE: Increases in hepatic and plasma cholesterol occur in patients with nonalcoholic fatty liver disease (NAFLD), although the reason for this is not well understood. We investigated whether Protease-Activated Receptor 2 (PAR2) plays a role in cholesterol and lipid homeostasis in NAFLD. METHODS: Human liver biopsies (n = 108) were quantified for PAR2 expression from NAFLD cases randomly selected and stratified by liver fibrosis stage, the primary predictor for clinical outcomes, while controlling for age, gender, and BMI between fibrosis groups. Demographic data and laboratory studies on plasma samples were obtained within 6 months of liver biopsy. Wild-type and PAR2-KO (C57BL/6 F2rl1-/-) mice were fed either normal or high fat diet for 16 weeks and plasma and liver assayed for lipids and soluble metabolites. RESULTS: Severity of NAFLD and plasma cholesterol levels significantly correlated with hepatocyte PAR2 expression in NAFLD patients. Conversely, PAR2 deficiency in mice resulted in reduced expression of key hepatic genes involved in cholesterol synthesis, a 50% drop in plasma and total liver cholesterol, and induced a reverse cholesterol transport system that culminated in 25% higher fecal bile acid output. PAR2-deficient mice exhibited enhanced fatty acid ß-oxidation with a ketogenic shift and an unexpected increase in liver glycogenesis. Mechanistic studies identified Gi-Jnk1/2 as key downstream effectors of protease-activated PAR2 in the regulation of lipid and cholesterol homeostasis in liver. CONCLUSIONS: These data indicate that PAR2 may be a new target for the suppression of plasma cholesterol and hepatic fat accumulation in NAFLD and related metabolic conditions.