Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic β-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca2+ permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca2+ permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat β-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca2+ measurements were performed using the fura-2 Ca2+ indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both β-cell lines and mouse islets. Yoda1 evoked Ca2+ entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from β-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca2+ and enhanced Yoda1 Ca2+ influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from β-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.

Pericytes regulate vascular development, stability and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFR β-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited peri-venous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in peri-venous and peri-arterial plexi and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the peri-venous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Rationale: A hallmark of type 2 diabetes is insulin resistance, which leads to increased endothelial cell (EC) production of superoxide and a simultaneous reduction in the availability of the vasoprotective signaling radical NO. We recently demonstrated in preclinical models that type 2 diabetes simultaneously causes resistance to IGF-1 (insulin-like growth factor-1)–mediated glucose lowering and endothelial NO release. Objective: To examine the effect of insulin and IGF-1 resistance specifically in ECs in vivo. Methods and Results: We generated mice expressing mIGF-1Rs (mouse IGF-1 receptors), which form nonfunctioning hybrid receptors with native IRs (insulin receptors) and IGF-1R, directed to ECs under control of the Tie2 promoter-enhancer. Despite EC insulin and IGF-1 resistance, mIGFREO (mutant IGF-1R EC overexpressing) mice had enhanced insulin and IGF-1–mediated systemic glucose disposal, lower fasting free fatty acids, and triglycerides. In hyperinsulinemic-euglycemic clamp studies, mIGFREO had increased glucose disposal and increased glucose uptake into muscle and fat, in response to insulin. mIGFREO had increased Nox (NADPH oxidase)-4 expression due to reduced expression of the microRNA, miR-25. Consistent with increased Nox4, mIGFREO ECs generated increased hydrogen peroxide (H 2 O 2 ), with no increase in superoxide. Treatment with catalase—a H 2 O 2 dismutase—restored insulin tolerance to WT (wild type) levels in mIGFREO. Conclusions: Combined insulin and IGF-1 resistance restricted to the endothelium leads to a potentially favorable adaptation in contrast to pure insulin resistance, with increased Nox4-derived H 2 O 2 generation mediating enhanced whole-body insulin sensitivity.

Introduction Ischemic stroke is commonly caused by large artery atherosclerosis. Patients with a high atherosclerotic burden (stenosis) and inflamed or ulcerated plaque are at increased risk of early recurrent ischaemic events. Treatment with anti-inflammatory agents may therefore reduce stroke incidence and recurrence in patients with this condition, but in vitro human studies of the possible mechanisms are lacking. We hypothesised that an anti-inflammatory compound (denoted drug X*) alters the expression of macrophage specific genes, including scavenger receptors in in vitro models of human carotid plaque macrophages. Methods CD14+ monocytes were collected from whole blood donations (volunteers) and differentiated to monocyte derived macrophages (MDMs). Macrophage polarisation with M1 (100 ng/ml LPS + 20 ng/ml INF-g) and M2a (20 ng/ml IL-4) markers was performed in complete media supplemented with 3 different clinically relevant concentrations of drug X.Gene expression (RT-qPCR) analysis (n=10) was carried out in the differentiated hMDMs to test for macrophage specific genes and scavenger receptors. Western Blot analysis (n=7) and immunofluorescence stain (IF) (n=5) was performed on drug X-treated MDMs to validate changes in the expression of Oxidized Low Density Lipoprotein Receptor 1 (OLR1). The physiological effect of drug X was also tested on oxLDL (25 mg/ml) uptake by macrophages (n=5). Results Our data suggest that drug X may re-program macrophages to a less inflammatory state (an M2 state) by significantly altering Mannose Receptor C-type 1 (MRC1) gene expression (p:0.0183). Drug X also alters the expression level of Macrophage Scavenger Receptor 1 (MSR1) gene, provoking the pacification of the M1 state and resulting in a beneficial effect on LDL uptake (p: 0.0845). Using OLR1 gene expression as a readout, in both M1 and M2a macrophages, the average OLR1 expression was reduced by treatment with drug X (p: 0.0003). This implies that M1 macrophages can bind less oxLDL which could reduce the formation of atherosclerotic plaque in the vessel wall. OLR1 protein expression was also reduced (p:0.0003) following 24-hour exposure of the macrophages to drug X. In functional tests, oxLDL uptake by drug X treated hMDMs was reduced by 44% (p:0.003). Conclusions The gene expression of macrophage specific genes and scavenger receptors in human M1 and M2a macrophages was significantly altered following 24-hour exposure to clinically relevant concentrations of drug X in vitro. Drug X reduced 'active' and pathogenic cell behaviours e.g., lipid uptake and promoted the polarisation to the M2 state.These data demonstrate that human macrophages can be reprogrammed to a less pathogenic state in vitro and they provide a potential mechanism for the effectiveness of drug X in the stabilisation of carotid atherosclerotic plaques in humans.

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