The Role of Endothelial PARs in Modulating Insulin Signaling

Abstract

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, Par1/4ECkomice 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 Par1/4ECkomice 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 Par4ECkomice 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.