Short-term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress

Abstract

To determine if short-term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium-dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 +/- 5 vs. 95 +/- 2% of maximum dilation, P < 0.05), whereas old calorie-restricted (OCR) and YCR did not differ (96 +/- 1 vs. 94 +/- 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 +/- 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium-independent dilation did not differ among groups. Short-term CR initiated in old age reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase-mediated superoxide production and stimulation of anti-oxidant enzyme activity), and upregulation of sirtuin-1.

Figure 1. Endothelium-dependent and -independent dilation in young and old calorie restricted (YCR and OCR) and ad libitum fed (YAL and OAL) B6D2F1 mice

Left: Dose-responses to the endothelium-dependent dilator acetylcholine (Ach) in the absence and presence of the endothelial nitric oxide synthase (eNOS) inhibitor N-G-nitro-L-arginine methyl ester (L-NAME) (YAL: n=5 YCR n=5, OAL: n=15 OCR: n=15). Right: Dose-responses to the endothelium-independent dilator sodium nitroprusside. Values are mean±SEM. * P<0.05 for main effect or interaction vs. other groups.

Figure 2. NO bioavailability and eNOS expression

Left: Nitric oxide-dependent dilation was calculated as the maximal dilation of carotid arteries to acetylcholine (ACh) minus the maximal response to ACh in the presence of L-NAME (Max Dilationach -Max Dilationach+l-name) in calorie restricted and ad libitum fed young and old mice (YAL, YCR, OAL, OCR). Right: eNOS protein in aorta expressed relative to GAPDH and normalized to YAL mean value. Representative blot shown. Values are mean±SEM, * P<0.05.

Figure 3. Oxidative stress

Left: Nitrotyrosine (NT) abundance in aorta of young and old calorie restricted (YCR, OCR) and ad libitum fed (YAL, OAL) mice assessed by western blot analysis. Data are expressed in relation to GAPDH and normalized to YAL mean value. *P<0.05 YAL vs. OAL and ^#P<0.05 CR vs. AL groups. Below: Representative blot shown. Middle: Mean electron paramagnetic resonance (EPR) signal from aortic rings (3 mm) showing superoxide production. *P<0.05 YAL vs. OAL and ^#P<0.05 CR vs. AL groups. Right: Maximal dilation of carotid arteries to acetylcholine (ACh) and to ACh + TEMPOL in the absence or presence of the endothelial nitric oxide synthase (eNOS) inhibitor N-G-nitro-L-arginine methyl ester (L-NAME) in YAL, YCR, OAL and OCR mice. *P<0.05 vs. ACh + TEMPOL and ^#P<0.05 vs. Ach within groups. Values are mean±SEM.

Figure 4. NADPH oxidase

Left: Enzyme activity and protein expression of NADPH oxidase (p67phox subunit) in aorta of ad libitum fed (AL) and calorie restricted (CR) young (Y) and old (O) mice (normalized to AL GAPDH controls). * P<0.05 vs. YAL group. Representative western blot shown below. Middle and Right: Endothelium-dependent dilation in the presence and absence of the NADPH-oxidase inhibitor apocynin in OAL and OCR (middle) and in YAL and YCR (right). P<0.05 for OAL vs. apocynin groups. Values are mean±SEM.

Figure 5. Manganese superoxide dismutase

Enzyme activity and protein expression of manganese superoxide dimustase (MnSOD) in aorta from young and old ad libitum fed (YAL and OAL) and calorie restricted mice (YCR and OCR). Values are mean±SEM, * P<0.05.

Figure 6. SIRT1

Protein expression of SIRT1 in aorta of young and old calorie restricted (YCR and OCR) and ad libitum fed mice (YAL and OAL) (normalized to YAL GAPDH controls). Representative blot shown below. Values are mean±SEM.* P<0.05.