Rajala, R. | bioRxiv Science

bioRxiv2025

A novel function for endothelial protease-activated receptors in modulating insulin receptor activity with implications for diabetes

Thrombin, a serine protease with increased activity in diabetics, signals through protease-activated receptors 1 and 4 (PAR1/PAR4) on endothelial cells (ECs). While studying the roles of endothelial PAR1/4 in diabetic pathology, we found that mice with inducible deletion of both receptors on ECs (Par1/4iECko) displayed increased insulin sensitivity and were protected against streptozotocin (STZ)-induced diabetes. Concordantly, we found that cultured primary ECs with PAR1/4 deficiency had increased basal activity/phosphorylation of the insulin receptor (IR) and insulin transcytosis. This elevated IR activity correlated with reduced activity of protein tyrosine phosphatase 1B (PTP1B), which is a negative regulator of IR activity. Lastly, Par1/4iECkomice with additional deletion of one allele of the IR gene demonstrated restoration of diabetic phenotypes after STZ treatment, indicating that these phenotypes are driven by heightened IR activity. These findings establish a novel link between endothelial PAR signaling and IR regulation, underscoring the critical role of ECs in metabolic homeostasis and identifying a potential therapeutic target for diabetes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/644607v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@4a5d4forg.highwire.dtl.DTLVardef@34012corg.highwire.dtl.DTLVardef@391efeorg.highwire.dtl.DTLVardef@21e3f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Acetaminophen Overdose Reveals Protease-Activated Receptor 4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium

Background & AimsHepatic endothelial cell (EC) dysfunction and centrilobular hepatocyte necrosis occur with acetaminophen (APAP) overdose. The protease thrombin, which is acutely generated during APAP overdose, can signal through protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity thrombin receptor that is known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generated during APAP overdose to determine (1) if hepatic endothelial PAR4 is a functional receptor, and (2) endothelial-specific functions for PAR1 and PAR4 in a high thrombin setting. MethodsWe generated mice with conditional deletion(s) of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte congestion/bleeding, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial Translating Ribosome Affinity Purification followed by next-generation sequencing (TRAPseq). ResultsWe found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability but no effect on hepatocyte necrosis. Additionally, mice with loss of endothelial Par1 and Par4 had reduced permeability at an earlier time point after APAP overdose when compared to mice singly deficient in either receptor in ECs. We also found that endothelial PAR1--but not PAR4--can regulate transcription in hepatic ECs. ConclusionsLow-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs. NOMENCLATUREF2r and F2rl3 are the gene names for PAR1 and PAR4, respectively. For simplicity, we hereafter refer to these genes as Par1 and Par4.

cell biology↗