Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs.

BACKGROUND: Sirtuins (SIRT) are NAD+-dependent deacetylases that are involved in stress response, antioxidative defense, and longevity via posttranslational modifications. SIRT1 directly activates nitric oxide synthase (NOS). Aging is associated with a reduced sirtuin function and reduction of the cofactor NAD+. Age-related atherosclerosis and vascular diseases are linked to a compromised sirtuin function. Vascular events like stroke and cardiac infarction result in acute hypoxia, which can additionally impact sirtuins and thus the vascular function. This prompted us to study sirtuins in intact HUVECs, under acute, short-term hypoxic conditions. METHODS: We measured intracellular sirtuin and NAD+ levels in HUVECs exposed to hypoxia (2% O2) for 10-120 min, compared to normoxic controls. SIRT1, SIRT3, and SIRT4 were measured at the protein (Western Blot) and the transcript level (qRT-PCR), SIRT1 and SIRT3 at the enzyme level (fluorometrically), and NAD+ levels were measured spectrophotometrically. RESULTS: We observed a reduction of SIRT1 and SIRT4 at the protein level, a downregulation of SIRT1 at the transcript level and increased NAD+ levels under hypoxia. SIRT3 was not affected by hypoxia. CONCLUSIONS: Downregulation of SIRT1 under hypoxia might reduce production of the reactive oxygen species (ROS) via the respiratory chain and inhibit the mitochondrial ATP-synthase, resulting in energy conservation. NOS might be impaired if SIRT1 is decreased. Increased NAD+ levels might compensate these effects. Hypoxic downregulation of SIRT4 might lead to mitochondrial uncoupling, hence endothelial dysfunction, and ADP/ATP-translocase 2 (ANT2)-inhibition. NAD+ upregulation might partly compensate this effect.

Impact of Nutrition on Short-Term Exercise-Induced Sirtuin Regulation: Vegans Differ from Omnivores and Lacto-Ovo Vegetarians.

Keywords: sirtuins; vegetarian; vegan; exercise; endurance athletes; metabolic regulation.

Alterations of sirtuins in mitochondrial cytochrome c-oxidase deficiency.

BACKGROUND: Sirtuins are NAD+ dependent deacetylases, which regulate mitochondrial energy metabolism as well as cellular response to stress. The NAD/NADH-system plays a crucial role in oxidative phosphorylation linking sirtuins and the mitochondrial respiratory chain. Furthermore, sirtuins are able to directly deacetylate and activate different complexes of the respiratory chain. This prompted us to analyse sirtuin levels in skin fibroblasts from patients with cytochrome c-oxidase (COX) deficiency and to test the impact of different pharmaceutical activators of sirtuins (SRT1720, paeonol) to modulate sirtuins and possibly respiratory chain enzymes in patient cells in vitro. METHODS: We assayed intracellular levels of sirtuin 1 and the mitochondrial sirtuins SIRT3 and SIRT4 in human fibroblasts from patients with COX- deficiency. Furthermore, sirtuins were measured after inhibiting complex IV in healthy control fibroblasts by cyanide and after incubation with activators SRT1720 and paeonol. To determine the effect of sirtuin inhibition at the cellular level we measured total cellular acetylation (control and patient cells, with and without treatment) by Western blot. RESULTS: We observed a significant decrease in cellular levels of all three sirtuins at the activity, protein and transcriptional level (by 15% to 50%) in COX-deficient cells. Additionally, the intracellular concentration of NAD+ was reduced in patient cells. We mimicked the biochemical phenotype of COX- deficiency by incubating healthy fibroblasts with cyanide and observed reduced sirtuin levels. A pharmacological activation of sirtuins resulted in normalized sirtuin levels in patient cells. Hyper acetylation was also reversible after treatment with sirtuin activators. Pharmacological modulation of sirtuins resulted in altered respiratory chain complex activities. CONCLUSIONS: We found inhibition of situins 1, 3 and 4 at activity, protein and transcriptional levels in fibroblasts from patient with COX-deficiency. Pharmacological activators were able to restore reduced sirtuin levels and thereby modulate respiratory chain activities.

HELLP Syndrome.

The hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is frequently observed in mothers whose offspring have long-chain fatty acid oxidation defects. We previously found that fatty acid oxidation is compromised not only in these inborn errors of metabolism but also in human umbilical vein endothelial cells (HUVECs) from all pregnancies complicated by the HELLP syndrome. Sirtuins are oxidized nicotinamide adenine dinucleotide (NAD+)dependent deacetylases linked to the metabolic status of the cell. SIRT 4 is known to have regulatory functions in fatty acid oxidation. The HELLP syndrome is often associated with short-term hypoxia. We studied sirtuins (SIRT 1, SIRT 3, and SIRT 4) in HUVECs from pregnancies complicated by the HELLP syndrome and uncomplicated pregnancies exposed to hypoxia (n = 7 controls, 7 HELLP; 0, 10, 60, or 120 minutes of 2% O2). Protein levels of SIRT 4 were significantly higher in HUVECs from HELLP compared to control after 60 and 120 minutes of hypoxia. The NAD+ levels increased in a time-dependent manner.