Restricting food availability to the active period restores rhythmic activation of aortic NOS3 in high fat diet fed mice

Abstract

Timing of food intake increases the risk for cardiometabolic disease. A chronic high fat diet (HFD) leads to endothelial dysfunction with loss of nitric oxide (NO) signaling in mice. Both endothelial function and vascular resistance display circadian variation with endothelial‐dependent relaxation and NO signaling peaking during the active period, while vascular resistance peaks shortly after waking. We hypothesized that HFD leads to a loss of circadian rhythm in endothelial NO synthase (NOS3) activation that is restored by restricting food availability to the active period. Mice (male 8‐week old, C57BL/6J) were given normal diet (ND; 10% fat) or HFD (45% fat) for 20 weeks ad libitum. Restricted feeding (RF; food availability only during the active phase) was enforced in half the mice during the final 2 weeks of feeding. Aortae were harvested at 4‐hour intervals over a 24‐hour period (ZT0 at 7AM; ZT12 at 7PM). NOS3 (total and phosphorylation at serine 1177 (pNOS3(S1177)) was assessed by Western blotting. NOS3 expression was unchanged with time in all 4 groups. In mice on ad libitum ND, pNOS3(S1117) expression displayed a diurnal variation with peak expression at ZT17. HFD mice displayed a significantly blunted amplitude and an earlier acrophase of the diurnal rhythm in pNOS3(S1117) expression by cosinor analysis (amplitude: HFD: 1.4±0.4 rdu; ND: 4.8±0.6 rdu, n=3, p=0.01; acrophase: HFD: 10.3±1.6 hrs, ND: 15.7±1.4 hrs, n=3, p=0.06). RF with HFD restored the acrophase of the pNOS3(S1177) rhythm (HFD: 10.3±1.6 rdu; HFD+RF: 16.4±1.4 rdu, n=3, p=0.04). To further examine the mechanism of NOS3 activation, we determined the expression of AMP kinase (AMPK), Akt kinase (Akt), and phosphorylation sites on each kinase at ZT5 and ZT17. Interestingly, RF in HFD mice significantly increased pAMPK(T172) abundance at ZT17 (n=4–5, p=0.02) compared to ad libitum HFD mice. Expression of AMPK, Akt, and Akt phosphorylation sites were unaffected by RF on ND or HFD. We further examined the 24 hour mean arterial pressure (MAP), heart rate (HR), and locomotor activity by telemetry. MAP was similar in all 4 groups (n=5–6, ND: 113±1 m Hg; ND+RF: 109±4 mmHg; HFD: 118±3 mmHg; HFD+RF: 114±2 mmHg). HR was increased with HFD and reduced in mice exposed to HFD+RF similar to ND mice (n=5–6, p<0.05). Locomotor activity was significantly decreased in HFD and HFD+RF mice compared to ND (n=5–6, p<0.05). In summary, aortic NOS3 activation displays a rhythm peaking during the active period at ZT17. HFD depresses the rhythm of aortic NOS3 activation, while RF restores the rhythm with an associated activation of AMP kinase at ZT17. We propose that time restricted feeding may lower the incidence of vascular disease through maintenance of rhythmic vascular NOS3 activation.Support or Funding InformationSupported by T32 DK007545 to PP, an AHA pre‐doctoral fellowship (18PRE33990345) to DZ, P01 HL069999 to JSP and DMP, and P01 HL136267 to JSP, EWI, and DMP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.