1. Investigación

Aim: In addition to functioning as an energy sensor switch, AMPK plays a key role in the maintenance of cardiovascular homeostasis. However, obesity disrupts AMPK signaling, contributing to endothelial dysfunction and cardiovascular disease. This study aimed to elucidate if a short-term dietary intervention consisting in replacing the high-fat diet with a standard diet for 2 weeks could reverse obesity-induced endothelial dysfunction via AMPK-CREB activation. Methods: For this, 5-week-old male C57BL6J mice were fed a standard (Chow) or a high-fat (HF) diet for 8 weeks. The HF diet was replaced by the chow diet for the last 2 weeks in half of HF mice, generating 3 groups: Chow, HF and HF-Chow. Vascular reactivity and western-blot assays were performed in the thoracic aorta. Results: Returning to a chow diet significantly reduced body weight and glucose intolerance. Relaxant responses to acetylcholine and the AMPK activator (AICAR) were significantly impaired in HF mice but improved in HF-Chow mice. The protein levels of AMPKα, p-CREB and antioxidant systems (heme oxygenase-1 (HO-1) and catalase) were significantly reduced in HF but normalized in HF-Chow mice. Conclusion: Improving dietary intake by replacing a HF diet with a standard diet improves AMPK-mediated responses due to the upregulation of the AMPK/ CREB/HO-1 signaling pathway.

Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphateactivated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation. Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5±0.05 nmol/mg vs. H2O2 levels fa/faCR=0.76±0.07 nmol/mg protein; p<0.05). Expression of mitochondrial superoxide dismutase (Mn-SOD) and total SOD activity were increased in aorta from fa/fa animals after CR. In cultured aortic endothelial cells, serum deprivation or 2-deoxy-D-glucose induced a significant increase in: i) superoxide anion and H2O2 levels, ii) p-AMPK/AMPK and p-eNOS/eNOS expression and iii) nitric oxide levels. This effect was reduced by catalase and strongly inhibited by Ca2+/calmodulin-dependent kinase II (CamkII) silencing. In conclusion, we propose that mild short-term CR might be a trigger of mechanisms aimed at protecting the vascular wall by the increase of H2O2, which then activates AMPK and nitric oxide release, thus improving endothelium-dependent relaxation. In addition, we demonstrate that CAMKII plays a key role in mediating CRinduced AMPK activation through H2O2 increase.