The Role of Endothelial PARs in Modulating Insulin Signaling

Endothelial cell (EC) dysfunction occurs in diabetes. Thrombin, a circulating serine protease with increased activity in diabetics, signals through the protease-activated receptors 1 and 4 (PAR1/PAR4). To determine EC-specific roles of PARs in insulin-dependent diabetes mellitus (IDDM), we generated Par1fl//fl ;Par4fl/fl ;iCdh5(PAC)-CreERT2 (Par1/4ECko ) mice, which we induced with tamoxifen to delete Par1/Par4 from ECs and subsequently challenged with streptozotocin (STZ) to induce diabetes. Diabetic Par1/4ECko mice had reduced blood glucose levels (347.6 mg/dl, p<0.001) compared to their diabetic control littermates (538.4 mg/dl). At 12 weeks after STZ treatment, Par1/4ECko mice also weighed significantly more than their diabetic control littermates (~6.5%, p<0.024). Par1/4ECko mice likewise had visible abdominal fat pads, which are normally lost during the progression of diabetes. In addition, serum albumin (Reference Range: 2.5-4.8 g/dl), which can be an indicator of hyperglycemia-induced dehydration, was significantly elevated in diabetic control mice (5.2 g/dl, p<0.0007) but attenuated in Par1/4ECko mice (4.46 g/dl). Importantly, diabetic Par1/4ECko mice did not have significantly different (p<0.4518) serum insulin levels than their diabetic control littermates. Therefore, since diabetic Par1/4ECko mice showed better glucose uptake parameters than their control littermates, despite comparably reduced insulin levels, the mutant mice have whole-body phenotypes that are consistent with heightened insulin sensitivity. Moreover, diabetic Par4ECko mice did not phenocopy the reduced blood glucose levels and weight loss seen Par1/4ECko mutants, indicating that endothelial Par1 deletion likely drives the heightened insulin sensitivity phenotypes. PAR1-siRNA-treated primary Human Umbilical Vein Endothelial Cells (HUVECs) showed increased basal insulin receptor (IR) activity (independent of insulin treatment) as measured by the uptake of a fluorescent glucose-analog. PAR1-siRNA-treated HUVECs also showed increased expression of the IR Type A (IR-A) spliceform, which has high insulin-independent basal activity. Conversely, treatment of HUVECs with a PAR1 ligand resulted in increased levels of the insulin-dependent IR Type B (IR-B)spliceform. These data suggest that endothelial PAR1 can act as a modulator of IR splicing/activity. IR activity in ECs is implicated in insulin transport from the bloodstream to parenchymal tissues. Increased endothelial IR signaling results in increased whole-body insulin sensitivity. We propose that endothelial PAR1 acts as a modulator of IR splicing/activity and subsequent insulin transcytosis. Therefore, loss of endothelial PAR1 potentially increases whole-body insulin sensitivity by driving insulin transport to parenchymal tissues and subsequent glucose uptake. © FASEB.