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Background: Systemic inhibition of mammalian target of rapamycin (mTOR) improves age-related arterial and metabolic dysfunction. Although the mechanisms and tissues involved in this amelioration remain unknown, endothelial cells (ECs), key regulators of arterial and metabolic function, may be responsible for these effects. Hypothesis: The beneficial effects of systemic mTOR inhibition will be recapitulated after EC specific mTOR deletion in old mice. Methods: We studied 22-24 mo old wildtype (WT) and EC specific tamoxifen-inducible mTOR knockout (KO) mice. The arterial function was determined by assessing aortic stiffness by measuring pulse wave velocity (PWV) using Doppler ultrasound as well as endothelium-dependent dilation (EDD) and nitric oxide (NO) bioavailability using pressure myography. Carotid artery superoxide production was measured using electric paramagnetic resonance. Glucose (2g/kg, ip) and lipid (3mL/kg, oral) tolerance tests were performed to examine metabolic function. Results: Two weeks after tamoxifen administration (4mg/day, 4 days, oral), we found a ~ 60% reduction in EC mTOR protein (p=0.04). Aortic PWV was lower in KO compared to WT mice (Fig A), indicating lower aortic stiffness. EDD to acetylcholine (ACh) was higher in mesenteric arteries from KO compared to WT mice (Fig B) due to an increase in NO bioavailability (31.6 ± 8.6 vs 8.4 ± 2.3; p=0.01). Superoxide scavenger, TEMPOL, increased EDD in both WT and KO mice (Fig C), eliminating differences between groups. Superoxide was lower in carotid arteries of KO compared to WT (Fig D), suggesting that oxidative stress impairs EDD in WT mice and ablation of EC selective mTOR ameliorates this effect. Aged KO mice demonstrated greater glucose and lipid tolerance compared to WT (Fig E, F). Conclusions: Our findings demonstrate that EC specific deletion of mTOR provides beneficial effects on arterial function via reductions in oxidative stress and prevents metabolic dysfunction in old mice.

Age‐related metabolic dysfunction is accompanied by accumulation of senescent cells and the senescence‐associated secretory phenotype (SASP) leading to chronic sterile inflammation. Recent evidence suggests that using a genetic approach to clear senescent cells improves metabolic function in old mice. However, the underlying mechanisms and if/how these findings can be translated into clinical practice are largely unknown. Here, we evaluated the impact of treatment with the FDA‐approved drugs, dasatinib and quercetin (D&Q), on tissue senescence burden, T lymphocyte infiltration and systemic metabolic function in 22‐24 month old C57Bl/6 mice. Administration of D&Q resulted in ~50% reduction in expression of the senescence marker p16 in perigonadal white adipose tissue (pgWAT) (p=0.004) and liver (p=0.003) (Fig 1A), but not skeletal muscle (p=0.76). D&Q treatment also resulted in 1 to 4‐fold reduction in expression of pro‐inflammatory SASP genes (i.e., mcp1, cxcl2, il‐6 and il‐1β) in pgWAT (all p≤0.04; Fig 1B), but not in liver and skeletal muscle (all p≥0.19), suggesting an adipose specific effect of D&Q. Using flow cytometry, we found that the reduced expression of p16 and SASP genes was associated with 30‐50% lower infiltration of total (CD3+), cytotoxic (CD8+) and helper (CD4+) T cells in pgWAT (all p≤0.04; Fig 1C). Treatment with D&Q ameliorated glucose tolerance (2 g/kg, ip) as demonstrated by a lower time response curve (group: p=0.007, interaction: p=0.03, Fig 1D) and reduced area under the curve (p=0.01, Fig 1E). To explore the underlying mechanisms, we assessed glucose‐stimulated (2 g/kg, ip) insulin secretion and insulin tolerance (1 U/kg, ip) and found no differences between groups (both p≥0.19). To assess the impact of D&Q on metabolic function in the liver, we performed a pyruvate tolerance (2 g/kg, ip) test and found that this treatment resulted in improved pyruvate tolerance (group: p=0.08, interaction: p=0.04, Fig 2A) and thus attenuated hepatic gluconeogenesis. Biochemical analysis revealed that phosphorylation of a gluconeogenic transcription factor, cAMP response element binding protein, and transcript level expression of gluconeogenic genes (pck1, pck2, fbp2 and g6pc) were lower in D&Q treated mice compared to vehicle treated mice (all p≤0.04; Fig 2B‐D), further supporting our findings of diminished hepatic gluconeogenesis. D&Q treatment also reduced plasma triglycerides (113 ± 5 vs 94 ± 5 mg/dL, p=0.02) and improved adipose tissue sensitivity to insulin as indicated by lower insulin‐stimulated ((1 U/kg, ip) plasma non‐esterified free fatty acids (fasted: 0.63 ± 0.02 vs 0.62 ± 0.06 mmol/L, p=0.87; 15 min: 0.54 ± 0.05 vs 0.33 ± 0.02 mmol/L, p=0.006). Taken together, these results suggest that D&Q improve glucose tolerance and lipid metabolism, associated with an attenuation of adipose inflammation (i.e., reduced SASP gene expression and T cell infiltration) and reduced hepatic gluconeogenesis in old mice.

Adenosine diphosphate (ADP) ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in numerous cellular processes including proliferation and membrane trafficking. Arf6 has been found to be dysregulated in diseases such as diabetic retinopathy and tumor metastasis. Furthermore, genetic insufficiency or pharmacological inhibition of Arf6 is protective in the aforementioned diseases, suggesting that Arf6 may be a potential therapeutic target. Here, we assessed the metabolic consequences of a reduction in Arf6 expression by performing glucose (GTT: 2 g/kg, ip), insulin (ITT: 1 U/kg, ip) and pyruvate (PTT: 2 g/kg, ip) tolerance tests in 3–4 mo old whole body Arf6 heterozygote (Arf6+/−) and wildtype (Arf6+/+) littermate control mice. Both protein and gene expression of Arf6 in the liver were 40–50% lower (p<0.05) in Arf6+/− mice compared to Arf6+/+. Body mass was higher in normal chow fed Arf6+/− compared to Arf6+/+ mice (30.3±0.9 vs 36.4±2.0 g, p<0.05). Blood glucose at 15, 30, and 60 min after injection during the GTT were 20–30% higher in Arf6+/− mice compared to Arf6+/+ (each p<0.05). Likewise, the area under the GTT time response curve was ~15% higher in Arf6+/− compared to Arf6+/+ (p<0.05), indicating impaired glucose metabolism. To examine the underlying cause of this glucose intolerance, we performed an ITT and assessed insulin‐stimulated suppression of plasma free fatty acids 15, and 30 min after insulin injection. We found no groups differences between the ITT time response curves (p>0.05) and no difference in the percent decrease in plasma FFAs from fasted between Arf6+/− and Arf6+/+ mice (~50% vs ~50% p>0.05). Likewise, fasted and glucose‐stimulated (2g/kg, ip) serum insulin were not different between the groups (2.44±0.58 vs 1.73±0.28, fasted; 4.54±1.74 vs 4.03±1.68 insulin stimulated; both p>0.05), suggesting that reductions in Arf6 expression do not impact pancreatic beta cell insulin secretion. Collectively, these measures suggest that neither peripheral insulin resistance nor impaired pancreatic beta cell function underlie glucose intolerance in Arf6+/− mice. To determine the contribution of hepatic gluconeogenesis to the observed glucose intolerance in the Arf6+/− mice, we performed a PTT and found that Arf6+/− mice exhibited a 15–25% increase in blood glucose over time compared to Arf6+/+ mice (p<0.05), suggesting a role for elevated hepatic gluconeogenesis. Taken together, this study demonstrates that Arf6 heterozygosity impairs glucose metabolism that is associated with aberrant hepatic gluconeogenesis but is independent of peripheral insulin resistance or pancreatic beta cell dysfunction. Support or Funding Information NIA R01 AG048366, R01 AG050238 and K02 AG045339, R01 AG060395 and US Department of Veterans Affairs I01 BX002151, I01 BX004492.

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 effec...