Ablation of Endothelial mTOR is Benign in Young Mice and Reverses Age‐Related Arterial and Metabolic Impairments in Old Mice

Abstract

Systemic inhibition of mammalian target of rapamycin (mTOR) improves age‐related arterial and metabolic dysfunction. Although the mechanisms and tissues involved remain unknown, endothelial cells (ECs), key regulators of arterial and metabolic function, may be responsible for these effects. In this study, we tested the hypothesis that EC mTOR will mimic systemic mTOR inhibition. To examine this, we studied young (4‐6 mo) and old (22‐24 mo) wildtype (WT) and EC specific tamoxifen inducible mTOR knockout (KO) mice. Arterial function was determined by assessing aortic stiffness by measuring pulse wave velocity (PWV) using Doppler ultrasound in vivo as well as endothelium dependent dilation (EDD) and nitric oxide (NO) bioavailability in isolated arteries using pressure myography. Carotid artery superoxide production was measured using electric paramagnetic resonance. Two weeks after tamoxifen administration (4mg/day, 4 days, oral), we found a 60‐70% reduction in EC mTOR protein (p=0.04). EC mTOR deletion did not alter PWV and EDD in young mice (Fig 1A, 1C), suggesting that EC mTOR is not critical to arterial function in young mice. We next sought to examine whether EC mTOR deletion reverses age‐related arterial dysfunction. Aging resulted in an elevated aortic PWV, indicating higher stiffness, in WT mice and deletion of EC mTOR attenuated aortic stiffening in old mice (p˂0.001, Fig 1A, 1B). Likewise, EDD to acetylcholine (ACh) was lower in mesenteric arteries in old WT mice compared to young mice (p˂0.01, Fig 1C, 1D). However, deletion of EC mTOR markedly improved EDD in old mice (Fig 1D) resulting from an increase in NO bioavailability (31.6 ± 8.6 vs 8.4 ± 2.3; p=0.012). The superoxide scavenger, TEMPOL, increased EDD in old WT and KO mice (Fig 1E), eliminating differences between groups. Furthermore, superoxide production was lower in carotid arteries of KO compared to WT mice (Fig 1F). Collectively, these results suggest that oxidative stress promotes arterial stiffness and impairs EDD with advanced age and ablation of EC selective mTOR reverses this effect. We next examined if this improvement in vascular function resulted in an improvement in metabolic function in the aged mice. To do so, we assessed glucose (2g/kg, ip), insulin (1U/kg, ip), pyruvate (2g/kg, ip) and lipid (3mL/kg, oral) tolerance. Aged KO mice demonstrated greater glucose and lipid tolerance compared to WT littermates (Fig 2A, 2D). Insulin tolerance and glucose‐stimulated insulin secretion did not differ between groups (Fig 2B, 2C), indicating that the improvement in glucose tolerance was independent of peripheral insulin sensitivity and pancreatic beta cell function. EC mTOR deletion improved pyruvate tolerance and reduced hepatic expression of gluconeogenic genes (Fig 2E, 2F), suggesting that attenuated hepatic gluconeogenesis may underlie the enhanced glucose tolerance in the KO mice. Taken together, our findings demonstrate that although without effect on vascular function in young mice, EC specific deletion of mTOR provides beneficial effects on arterial function via reductions in oxidative stress and ameliorates metabolic dysfunction in old mice.